Final Report Summary - EUCOMMTOOLS (EUCOMM: Tools for Functional Annotation of the Mouse Genome)
Executive Summary:
Part I: EUCOMMTOOLS Executive Summary
The EUCOMMTOOLS (The European Conditional Mouse Mutagenesis TOOLS) Consortium executes conditional gene targeting in murine embryonic stem (ES) cells and establishes Cre-driver transgenic lines for the determination of spatial and temporal gene function.
EUCOMM (FP6) and EUCOMMTOOLS (FP7) are founding members of the International Knockout Mouse Consortium (IKMC, www.knockoutmouse.org) aiming to establish gene mutations for all 20.000 protein coding genes which will build the basis of gene function annotation at scale. The major objective of EUCOMMTOOLS was to complete the conditional gene targeting resource, to establish Cre-driver lines, to further develop conditional tools, and to extend the genetic tool kit for universal use of EUCOMM/EUCOMMTOOLS targeted alleles.
Towards these goals EUCOMMTOOLS has established highly standardized pipelines to reproducibly establish targeting vectors, mutant ES cells and mutant mice under stringent quality control conditions.
In particular EUCOMMTOOLS has established: 1) 2.957 correctly conditionally targeted ES cells, 2) a new conditional targeting strategy for single exon genes, 3) an efficient CRISPR/Cas9 mediated targeting strategy to generate complex conditional alleles, 4) novel universal genetic tools for ectopic gene expression, fluorescence protein tagging, and cell ablation, 5) the evaluation of the risks of site specific mutagenesis approaches, and 6) 223 novel Cre-driver lines and determined partly their Cre-activity.
To date, in a combined effort about 18.500 gene mutations in ES cells have been established. The majority of mutant ES cell lines (about 13.000) have been generated by EUCOMM/EUCOMMTOOLS programs. Based on this resource the International Mouse Phenotyping Consortium (IMPC) has already established approximately 5.000 mutant mice which are currently being phenotyped. The common goal of IKMC and IMPC is to establish an encyclopedia of gene functions serving as reference for the functional annotation of any disease associated allelic variant. The gene targeting vectors and mutant ES cells of EUCOMM/EUCOMMTOOLS are archived by the European Mouse Mutant Cell Repository (EuMMCR, www.eummcr.org) and the mice by EMMA/Infrafrontier (www.infrafrontier.eu). Up to now, more than 1.000 conditional targeting vectors, more than 12.000 mutant ES cells, and more than 1.800 mutant mice have been distributed to academic and commercial users worldwide. Furthermore, more than 1.000 publications acknowledge the use of EUCOMM/EUCOMMTOOLS materials already (source: google scholar).
The success of this ground breaking European programs goes beyond its funding period as EUCOMM/EUCOMMTOOLS have established a self-sustaining repository; the EuMMCR (www.eummcr.org) providing the generated material to academic and commercial researchers also in the future.
Project Context and Objectives:
Part II: EUCOMMTOOLS Context and Objectives
One of the most exciting and valuable scientific challenges is the elucidation of gene function in the context of the entire organism. This elucidation will bring us as close as never before to the understanding of mammalian biology and offer the possibility to fight suffering and disease more efficiently than ever.
Certainly, such an ambitious goal can only be reached in steps. The decoding of the human and mouse genome sequences has been a very important milestone, which resulted in the identification of approximately 20.000 protein coding genes as well as an astonishingly high number of conserved non-coding regions with assumed regulatory functions. The next logical step is to functionally annotate the genome.
This challenge can only be met by comparing gene functions in healthy and hereditarily diseased organisms. Due to embryonic stem (ES) cell technologies, any desired gene mutation can be established in the laboratory mouse, so that all hereditary human diseases can be modeled in this organism. In addition, recent technology developments now allow performing gene mutagenesis in a systematic, high throughput manner to eventually mutate every gene within the mouse genome. This enormous task can only be achieved by an international, coordinated effort, because such a systematic approach will reduce research redundancy, facilitate standardization, and enable the global implementation of technical developments.
In 2007, a worldwide mutagenesis effort began to form which aims at mutating virtually all protein coding mouse genes via gene targeting and gene trapping to speed up functional annotation of the genome. The cornerstones of this International Knockout Mouse Consortium (IKMC) are the European EUCOMM/EUCOMMTOOLS, the U.S. KOMP and the Canadian NorCOMM projects (Collins F.S. et al., 2007a; Collins F.S. et al., 2007b; Skarnes et al., 2011; Bradley et al., 2012; Murray et al., 2012; Donahue et al., 2012, Rosen et al. 2015; www.knockoutmouse.org). The European flagship EUCOMM/EUCOMMTOOLS contributes the largest fraction of mutant mouse genes to the IKMC. Furthermore, EUCOMM/EUCOMMTOOLS was the first project to recognize the tremendous value of conditional mutagenesis (Averx et al., 2004). Based on IKMC, the International Mouse Phenotyping Consortium (IMPC) has been founded to turn the IKMC mutant ES cell resource into mutant mice and determine the mutant phenotype at scale establishing an encyclopedia of gene function. This resource will serve as reference for future disease models.
EUCOMMTOOLS will complete the IKMC resource of mutations for all protein coding genes as far as technically possible. Furthermore, EUCOMMTOOLS will maximize the utility of the conditional IKMC resource by generating up to 250 different, mostly inducible Cre driver mouse lines. In addition, EUCOMMTOOLS will develop novel tools to enhance the versatility of the IKMC resource. EUCOMM’s and EUCOMMTOOLS’ vectors and mutant ES cells are distributed worldwide via the EuMMCR (www.eummcr.org) and the projects’ mice via EMMA (www.emmanet.org). The organizational structure of the EUCOMMTOOLS project is shown in Figure A.
The detailed objectives of the EUCOMMTOOLS program are:
a) to identify 500 genes and their promoters with specific tissue and cell type expression profiles suitable as Cre drivers covering all organs and the major cell types of the body,
b) to establish up to 500 Cre Knockin constructs and recombinant ES cell lines,
c) to establish up to 250 BAC-CreERT2/BAC-Cre driver transgenic mouse lines, and to annotate Cre expression and activity of all 250 Cre driver lines in tissue sections to determine organ and cell type specificity,
d) to provide ‘null mutant first conditionally suitable’ mutant ES cells of approximately 500 miRNA loci. This specific aim was already performed by the Wellcome Trust Sanger Institute, supported by institutional funds. The mutant ES cells have been integrated into the EUCOMMTOOLS resource and are distributed by EuMMCR,
e) to develop gene cassettes for integration into the conditionally mutated EUCOMM alleles such as: cassettes introducing any open reading frame (ORF) of interest, various reporter genes, suicide genes, various recombinases such as Flp, Dre, Cre-ERT2; or siRNAs. These cassettes can easily be integrated into the conditional EUCOMM alleles, either by Recombinase Mediated Cassette Exchange (RMCE) in ES cells, or by TALEN- or CRISPR/Cas-mediated homologous replacement in oocytes,
f) to improve recombinases: to investigate ligand-receptor pairs to improve specificity of recombinases; to develop sequential, conditional switches; to determine position effects of recombination target sites; and to determine recombinase toxicity,
g) to strive towards completion of the IKMC resource by generating up to 3.500 unique, conditionally gene targeted mouse ES cells.
h) to present all EUCOMMTOOLS materials to the public via the EUCOMMTOOLS and IKMC/IMPC web portals,
i) to archive and distribute EUCOMMTOOLS materials via EuMMCR and EMMA,
j) to sustain the EUCOMMTOOLS/EUCOMM and IKMC resource in the long term.
The “Large-scale Integrating Project” EUCOMMTOOLS brings together nine research institutions from five European countries, Australia and USA: the Helmholtz Zentrum München, Munich/Neuherberg, Germany (Prof. Wolfgang Wurst, (EUCOMMTOOLS coordinator), Dr. Joel Schick, Dr. Ralf Kühn (until 06/2014), Dr. Antje Bürger); the Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK (Prof. Allan Bradley, (EUCOMMTOOLS co-coordinator), Dr. William Skarnes, Dr. Vivek Iyer (until 01/2015), Dr. Ramiro Ramirez-Solis); the European Molecular Biology Laboratory (EMBL), Outstation EBI Hinxton, UK (Dr. Paul Flicek, Dr. Gautier Koscielny (until 09/2014), Dr. Terry Meehan); the University of Technology Dresden, Germany (Prof. Francis Stewart, Dr. Konstantinos Anastassiadis); the Consiglio Nazionale delle Ricerche, Monterotondo, Italy (Prof. Glauco Tocchini-Valentini); the Institut Clinique de la Souris, Strasbourg, France (Prof. Yann Hérault, Dr. Marie-Christine Birling); the Universidad de Murcia (Prof. Salvador Martínez); the Medical Research Council (MRC), Mammalian Genetics Unit, Harwell, UK (Prof. Steve Brown), and Human Genetics Unit, Edinburgh, UK (Prof. Richard Baldock), Monash University (EMBL Australia), Melbourne (Prof. Nadia Rosenthal) and the Jackson Laboratory (JAX), Bar Harbor, USA (Dr. Steve Murray). These institutions bring together skills, expertise, resources, and infrastructure to fulfil the ambitious EUCOMMTOOLS goals.
Project Results:
PART III: EUCOMMTOOLS Scientific and Technical Results/Foregrounds
WP1 Gene Selection
WP Leader: N. Rosenthal (MONASH), Partners: A. Bradley (WTSI), Y. Herault (ICS), W. Wurst (HMGU), R. Baldock (MRC/HGU), P.Flicek/G. Koscielny, T. Meehan (EMBL-EBI)
Cre driver selection
WP1.1 has achieved all its tasks. It continued collecting data on publicly available Cre recombinase expressing transgenic mouse lines and presenting them at the CREATE/EUCOMMTOOLS portal (www.creline.org). It also further monitored and incorporated scientists’ suggestions of novel genes for Cre driver production using this portal. For international coordination of Cre driver generation activities, in the last reporting period the Jackson Laboratory was integrated as partner. To avoid duplicated efforts in Cre mouse production, the CREATE website was regularly updated to show the planned and produced resources of the respective Cre driver production projects worldwide including EUCOMMTOOLS. Based on various selection criteria, WP1.1 has identified 732 genes which are suitable for the establishment of specific Cre driver lines. In particular, genes were excluded leading to haploinsufficiency when mutated which is an important aspect when employing knockin strategy (see WP2.1). This list is also presented at the CREATE/EUCOMMTOOLS web portal, indicating also the current Cre driver production status. A regularly updated interface on the CREATE website that displays the produced resources of all the EUCOMMTOOLS Cre lines was created. This is an automatically updated production tracker for each Cre line: As the mice get produced, the production tracker is updated. In addition, a feature was added to the website that provides links from a gene driver to the resulting expression data annotation resource being generated as part of WP6. We have also put in place the necessary informatics infrastructure needed to analyze and display phenotype data from WP7 that will be presented on the IMPC portal: mousephenotype.org.
Gene selection for targeting
The gene list for the EUCOMMTOOLS gene targeting pipeline (WP1.2) contains 3.600 remaining bona fide annotated protein-coding genes. Designs have been generated for all 3.600 genes based on the conventional EUCOMM conditional allele or the artificial intron strategy. Alternative designs have been established for 1.400 genes that failed to produce a conditional targeting vector in the EUCOMM/KOMP-CSD pipelines. Thus, all proper annotated protein-coding genes went through the EUCOMMTOOLS vector production pipeline.
WP2 Vector Construction
WP Leader: W. Skarnes (WTSI), Partners: A. Bradley (WTSI), F. Stewart (TUD)
Generation of conditional KO-first targeting constructs
Since the EUCOMM conditional targeting vectors cannot be used for single exon genes, in WP2.2 a novel strategy was developed by integrating an artificial intron fragment into the conditional gene targeting cassette in the framework of EUCOMMTOOLS. The intron sequence is derived from the Ifitm2 gene, a two exon gene that is highly expressed in all mouse tissues. EST alignments show no evidence for intron retention or cryptic splice sites and the intron is devoid of any conserved elements based on cross-species sequence comparison. A knockout-first allele targeting vector was generated for this gene by KOMP-CSD, and thus we were able to subclone a fragment from the intermediate vector that contains the Zeo-PheS Gateway element imbedded in the intron (see Figure 1). The Ifitm2 gene was cloned by gap repair into a R6K plasmid backbone to generate the template for recombineering (pR6K_Ifitm2i_ZP). To introduce the artificial intron cassette into a BAC, 70-mer oligos are synthesized that contain 20 bp from the 5’ or 3’ end of the intron appended to 50 bp of sequence from either side of the insertion point in the exon of the target gene.
Figure 1. (see attachment)
To validate the artificial intron strategy, we generated targeting constructs for 4 X-linked genes expressed in ES cells (Atrx, Mecp2, Hdac8, and Glp3) by BAC recombineering. The final targeting constructs contain the standard promoterless EUCOMM cassette. Targeted clones were then sub-cloned to ensure they were pure clones and analysed by Western blot for protein levels with commercial antibodies. Only the Atrx gene gave a good signal on a Western, whereas Mecp2 gave a weak signal and no protein was detected with Hdac8 and Glp3 antibodies. The Atrx cells were then transiently transfected with a Flpo expression plasmid to remove the targeting cassette from the artificial intron. As shown in Figure 2, insertion of the artificial intron cassette completely eliminates Atrx protein expression, whereas removal of the targeting element with Flp restores wild-type levels of Atrx protein. These experiments show that the artificial intron strategy is working as desired.
Figure 2. (see attachment)
Corresponding to the new design, quality control (QC) strategies have been developed to validate each step in vector generation. During the whole duration of the project a total of 2.339 targeting vectors have been generated, and recovery attempts were underway for 3.500 projects where a vector is available but electroporation had not been successful enough, i.e. for which less than three ES cell clones are available.
CRISPR/Cas9-assisted targeting
CRISPR/Cas9 sRNA-guided endonucleases may offer an efficient strategy for the recovery of failed conditional targeting projects (Figure 3). Computational analysis has identified suitable CRISPR sites at the 3’ loxP site of the targeting vector for 90% of the failed projects.
Figure 3. (see attachment)
Over the past year, we generated gRNA plasmids for 88 failed genes and tested the efficiency of Cas9-assisted targeting as a recovery strategy. We reasoned that many of the failed projects may be due to difficulties with LR-PCR genotyping across the long homology arms, rather than a failure to target the desired locus. Therefore, we have also developed a process to shorten the vector arms based on linear:linear ET cloning recently described by Francis Stewart (WP3) and colleagues (Fu J et al., Nature Biotechnology 2012).
Vector Shortening Process to Facilitate Cas9 Assisted Targeting Route
Conventional conditional vector targeting failures in the main EUCOMMTOOLS pipeline can be due to a combination of two problems, low targeting frequency and difficulties in LR PCR genotyping. While Cas9-assisted targeting can address the former issue, it does not help resolve the latter. As Cas9-assisted targeting requires much shorter targeting arms (1kb or less) than the conventional route, it would also be optimal to shorten the 5kb targeting arms found in EUCOMMTOOLS vectors to 1kb or less to facilitate genotyping and improve its success rate. To that end, we have developed a high throughput process for shortening the arms of EUCOMMTOOLS conditional gene targeting vectors in a 96 well format (Figure 4). The process is based on linear-linear recombineering using the recET recombineering system (Fu et al., Nature Biotechnology 2012).
Figure 4. (see attachment)
We have this process operating in a 96 well format and, to date we have generated 242 shortened vectors. These vectors, along with the corresponding gRNA plasmids, have been transferred to WP4.2 for ES cell production.
In summary, from 3.750 gene designs we have successfully constructed 2.339 targeting vectors including 330 vectors for CRISPR/Cas mediated targeting. In addition 1.679 vectors were recovered from failed project. Thus in total we have provided 4.018 vectors for electroporation in ES cells.
Cre-BAC knock-in vectors for recombination-mediated cassette exchange at a pre-defined locus
The goal of WP2.1 was to construct the vectors for 500 BAC-CreERT2/BAC-Cre transgenic ES cell lines. The project’s primary approach towards the objective of this work package was to use recombinase mediated cassette exchange (RMCE) as a means to integrate single-copy BAC-Cre recombinase transgenes at pre-determined loci (ROSA26 and HPRT) in ES cells. It was previously reported that for the site specific integration of BAC transgenes the efficiencies of transgenesis were too low to be used for pipeline production. During pilot studies the HPRT landing pad integration failed to yield any BAC integrants and the ROSA26 landing pad integration produced very low rates of integrated BACs.
The low efficiencies of site specific BAC transgenesis in the C57BL/6N ES cells that we have experienced led to the suggestion that an alternative approach might be used. In this alternative approach the BAC would be modified by insertion of piggyBac transposon inverted repeats within the BAC vector backbone in close proximity to the genomic DNA cloning sites. It has been demonstrated that by pronuclear injection of a mixture of piggyBac transposase mRNA and the modified BAC a high level of single copy, randomly integrated BAC transgenes ca be achieved in single cell embryos, which can then develop to make transgenic mice. This technology allows the direct production of transgenic offspring, circumventing ES cell and chimera generation.
The gene specific modifications of the BAC inserting the EGFP-CreERT2 are performed exactly as for the previous (site specific integration) pipeline. Following the gene-specific modification the production pipelines diverge as there are two further recombineering steps that insert piggyBac 5’ and then 3’ inverted repeats at either ends of the BAC vector backbone.
The BAC constructs have been sent to mouse production centres, to generate transgenic offsprings. After generating 15 transgenic lines it turned out that the vector was not stable as shown by restriction enzyme mapping and finally by DNA sequencing. Therefore, this strategy was abandoned.
As an alternative strategy for the BAC-CreERT2 approach, in parallel we have developed a working pipeline for the construction of Cre recombinase knockin targeting vectors involving the re-cycling of intermediate vectors made for IKMC projects (EUCOMM, EUCOMMTOOLS, KOMP) exploiting their modular Gateway capacity. No new recombineering based vector construction is involved, just a single high efficiency vector assembly step. The major components, a gene specific intermediate vector and a reading frame specific nlsGFP/CreERT2 knockin cassette are shown below (Figure 5):
Figure 5. (see attachment)
Intermediate vectors are re-arrayed onto 96 well plates, and their identity is confirmed by DNA sequencing with a >95% success rate. The resulting validated intermediate vectors are then put through a three way Gateway reaction (intermediate vector, Cre cassette and DTA containing plasmid backbone), and multiple colonies are analysed by DNA sequencing. The integrity of the resulting Cre knockin final vectors is confirmed by a combination of physical (gel of linearised plasmid DNA) and DNA sequencing (gene identity, reading frame of CreERT2 cassette). The efficiency of this process was 100% with 457 final vectors assembled to date. Vectors that failed targeting with the PGK-puromycin version were reassembled with the Beta-actin neo cassette (which targets at a higher success rate) to rescue the projects. Successful clones were re-arrayed onto 96 well plates for electroporation into JM8 ES cells.
Furthermore, within this workpackage, the EUCOMMTOOLS consortium has commenced a pilot experiment Figure 6) to test the possibility of expedited engineering of Cre knockin mice directly in mouse zygotes exploiting the CRISPR/Cas9 system. This would create an alternative and speedy route to Cre knockin mice for the Cre knockin mouse generation project.
Figure 6. (see attachment)
Vector shortening for CRISPR-assisted targeting in mouse zygotes
In cases where CreERT2 knockin vectors have not yielded efficient targeting in ES cells even after repeated electroporations there is a possibility of rescuing the project by the Cas9-assisted targeting route in mouse zygotes. However, to optimize the probability of success of this strategy the following modifications of the targeting vectors must be performed:
1) the vector must not contain gRNA recognition sites so it should not be cleaved by Cas9 nuclease
2) shortening the targeting arms from 5kb to 1kb should improve genotyping
3) CreERT2 cassettes no longer requires a mammalian drug selection marker
Employing the linear-linear ET recombination vector shortening process described in the WP2.2 section, the intermediate vectors for 48 EUCOMMTOOLS Cre genes which have definitively failed targeting are being converted to short arm vectors suitable for Cas9-mediated targeting in mouse zygotes (Figure 7).
Figure 7. (see attachment)
The intermediate vectors that enter the process have been previously sequence validated and only require re-arraying to commence the vector shortening process. Pairs of U6 gRNA plasmids with minimal off target signatures to sequences in the deleted genomic region are also produced. A version of the standard EGFP_CreERT2 cassette in which the mammalian selection marker has been deleted by exposure to Dre recombinase in Dre expressing E. coli is used for the Gateway vector assembly reaction.
CRISPR/Cas9-Assisted Targeting directly in mouse zygotes for making Cre Knock-in mice
In collaboration with Steve Murray’s group at the Jackson laboratory, we were building 48 short arm targeting vectors tailored for use in mouse zygotes using a Gibson assembly strategy. This route can be used to construct alternate alleles such as 5’ ATG knock-ins where the endogenous start codon (ATG) is replaced with a Kozak ATG version of the same EGFP_T2A_CreERT2 cassette used in the EUCOMMTOOLS high throughput Cre knockin pipeline. There is no need for a selection marker cassette for vectors destined for direct injection into the mouse zygote. The Gibson assembly vector assembly strategy shown in Figure 8 combines:
1) PCR amplification of 1 kb targeting arms from C57BL/6 genomic DNA
2) a custom R1R2 Gateway Cassette with Zeocin resistance
3) an Amp resistant plasmid backbone
Figure 8. (see attachment)
Thus, this technology paves the way for next generation Cre-driver line production.
In summary, we have generated 55 BAC- CreERT2 vectors, 447 CreERT2 Knock-in vectors and 12 short arm CreERT2 Knock-in vectors for CRISPR/Cas9 mediated targeting in zygotes. In total, we have produced 514 CreERT2 targeting vectors.
WP 3: Research, Technology and Reagent Development
WP Leader: F. Stewart (TUD), Partners: A. Bradley (WTSI), R. Kühn (HMGU), W. Skarnes (WTSI), W. Wurst (HMGU)
Establishment and validation of an IKMC toolkit
The focus of WP3 was the development of new technologies to optimize the utility of the conditional EUCOMM/TOOLS and IKMC resources. In particular, universal cassettes (for reporters such as YFP and LacZ and recombinases such as EGFP-CreERT2) have been produced (WP3.1).
Construction of a universal cassette for retargeting/RMCE.
We have developed a series of dual-purpose universal cassettes for the introduction of fluorescent proteins and a variety of other reporters/recombinases into EUCOMM(TOOLS)/KOMP genes by facilitated gene targeting or by recombination-mediated cassette exchange (RMCE). Each of these constructs is flanked with attL1/L2 sites such that they can be assembled into targeting vectors in an in vitro Gateway reaction with Gateway-adapted intermediate vectors from the EUCOMM(TOOLS)/KOMP resource (see Figure 9).
Figure 9. (see attachment)
The universal cassettes contain site-specific recombination sites (5’ Frt and 3’ loxP) for dual recombinase dRMCE (Osterwalder M., et al. (2010), Nat Methods 7(11):893-5) in targeted ES cell clones. Expression of the reporter is dependent on splicing and the production of a fusion protein. Therefore, we engineered these cassettes in each of the three reading frames as well as a Kozak-ATG version for expression from non-coding exons. For optimal expression of the reporter, we included a novel 2A-like sequence (LF2A) which our experimental evidence (presented in last year’s report) suggests can be used efficiently in fusions to both secreted/transmembrane and non-secreted proteins.
Construction of a universal targeting cassette.
The activity of the lacZ gene is compromised in fusions to genes that encode N-terminal signal sequences (Skarnes WC, et al. Proc Natl Acad Sci USA. 1995 Jul 3;92(14):6592-6). For the EUCOMM project, we used a promoter-targeting cassette that contains an internal ribosome entry site (IRES) to initiate translation of the lacZ reporter. Expression of lacZ is frame independent and should work irrespective of protein class. However, efficient use of the IRES sequence is known to be influenced by local sequence content and thus may not be reliable in all genes. To explore the L2A sequence further, we constructed promoter-driven targeting cassettes that contain this sequence fused to the 5’ end of lacZ (see Figure 10) and carried out gene targeting experiments for several non-secreted and secreted loci. In these experiments, we included promoter-driven targeting cassettes that contain the IRES sequence for comparison. Based on the intensity of Xgal staining in cells, the LF2A cassette showed equal or higher levels of staining for both secreted and non-secreted targeted loci compared to the IRES cassette. Now reading frame specific LF2A targeting cassettes are routinely used in the construction of most EUCOMMTOOLS conditional targeting vectors
Figure 10. (see attachment)
Construction of Cre cassettes.
At the EUCOMMTOOLS meeting in Alicante in April 2011, it was agreed to pursue Cre knock-ins into a selected set of target genes and to compare this method to RMCE of Cre-BACs into Hprt and Rosa26. Subsequently it was decided to develop a full scale production pipeline of targeted Cre knockins described in WP 2.1. To this end, and with input from the EUCOMMTOOLS Scientific Advisory Board, we developed dual eGFP reporter/inducible Cre recombinase (CreERT2) cassettes that are used for knock-in strategies, primarily re-targeting of Gateway assembled pre-existing intermediate vectors that also can be used for dRMCE. Co-expression of eGFP provides a cellular marker for CreERT2 expression, facilitating the characterisation of Cre protein expression by direct fluorescence or by immunohistochemistry with anti GFP antibodies. The vectors now used are shown in Figure 11. Initially a PGK puromycin selection cassette version was developed for RMCE (standard conditional IKMC cells are neoR). However, these vectors delivered less efficient targeting compared to standard EUCOMMTOOLS conditional alleles, so an alternate Beta-actin-neo version was built and is now used in the Cre Knock-in targeting pipeline.
Figure 11. (see attachment)
The functionality of tamoxifen inducible Cre activity and fluorescent protein expression was tested in an ES cell line containing a floxed lacZ reporter in a ubiquitously expressed gene (Arid1a).
The first crosses of Cre knockin mice for the secreted Gpx3 indicate robust, tamoxifen inducible, activity of Cre on a floxed lacZ reporter in the ROSA 26 locus in vivo, demonstrating the full functionality of the vector in vivo.
A vector for the knock-in of EGFP_2A_CreERT2 into the 3’ UTR of endogenous genes (IRES/nlsEGFP/CreERT2) has also been developed. This vector should avoid potential haploinsufficiency associated with knock-in alleles. De novo recombineering/Gateway Vector assembly for a set of 24 genes resulted in 19 final IRES_nlsEGFP_CreERT2 3’ knockin vectors which have been targeted in ES cells. As this route involves new recombineering of targeting vectors, it does not represent a solution for accelerating the production of EUCOMMTOOLS Cre knockin mice, but the alleles are of interest as a comparison to other Cre knockin strategies and for cases of potential haploinsufficiency.
Other EUCOMMTOOLS cassettes.
EUCOMMTOOLS cassettes have been classified into cassettes which can be used with existing off-the-shelf IKMC intermediate vectors, 3’ IRES containing vectors for insertion after the termination codon of the endogenous protein and 5’ KATG (Kozak-ATG) cassettes used to replace the initiating methionine; the latter two approaches require custom recombineering approaches utilizing EUCOMMTOOLS vector components. Modular cloning approaches allow re-cycling of validated vector components into these 5’ and 3’ cassettes. The list includes cassettes for a variety of fluorescent proteins (presently being incorporated into the modular vector system are far red, orange and blue fluorescent proteins), chromosome engineering that allow fluent replacement of rearranged sequences, multimodal reporters employing 2A sequences (fluorescent, luminescent, MRI), and neutral surface reporters for isolating cell populations (developed in collaboration with Timm Schroeder, HMGU Munich), and a 3’ protein tagging system.
Table 1. (see attachment)
One of the cassettes we have developed is a tri-modal reporter which combines fluorescence, luminescence and PET imaging (HSVtk) reporters as shown in Figure 12. It will be employed to track transplanted mouse and human stem cells in animal models of human diseases. All three reporter activities have been validated in mouse ES cells with CAGGS PiggyBac integrations of the triple reporter in collaboration with Sanjay Sinha’s group at the Laboratory of Regenerative Medicine, Cambridge.
Figure 12. (see attachment)
Several of the EUCOMMTOOLS cassettes have also been validated by gene targeting human ES cells (H9 and H1) in collaboration with Prof. Roger Pedersen’s laboratory in Cambridge and thus are also of value in engineering human as well as mouse stem cells.
We have also developed a new cassette which has been employed in the conditional disruption of long non-coding RNAs (lncRNAs) in collaboration with Prof Adrian Bird’s laboratory in Edinburgh. This cassette contains elements to truncate both spliced and unspliced lncRNA transcripts using Rabbit beta-globin exon/intron and repeated SV40 polyA sites with site specific recombination sites to enable a conditional strategy which involves either deletion of the promoter or truncation of the transcript. It has now been employed in constructing CKO vectors for more than 50 lncRNA targets. The disruption cassette’s structure is shown below Figure 13):
Figure 13. (see attachment)
The development of neutral ligands for mammalian SSR-LBD applications
To improve the function of the CreERT2 estrogen-ligand binding domain, a mutant library of estrogen binding domains (Cre-EBD) has been created. Its screening protocol has been optimized (WP3.2). We decided, however to prioritize WP3.3 to enhance Cre-driver production first.
Cre-EcBD (ecdysone BD) fusion proteins were built and functionally evaluated. Previously the Stewart lab evaluated the ecdysone BD in similar experiments to conclude that the EcBD did not convey repression onto Cre recombinase in the absence of ligand (unpublished results). Further experiments, performed in ES cells with a positive control conclusively reinforce the previous conclusion – the EcBD does not inhibit fusion proteins in cis. Hence we discontinued EUCOMMTOOLS experiments with the EcBD. The development of a new recombineering method for codon-directed, site-specific mutagenesis has been successful. Using this method, a very high quality site directed mutant library of the estrogen binding domain (EBD), with codon-based mutations focused on 36 amino acids around the ligand binding pocket, has been made. Furthermore, the yeast shuttle vector for expression of the mutant EBD library has been finalised. A pilot experiment with a FLP-wtEBD showed satisfactory properties of recombinase repression before, and recombinase activity upon, ligand induction. After removal of a restriction site, the final yeast shuttle vector for the molecular evolution experiments has been completed. Pilot experiments in yeast with FLP-wtEBD encountered problems with low transformation efficiency. This bottleneck has now been solved and high transformation efficiency has been obtained to introduce more than 10.000 mutant EBD clones into yeast for selection. We have established mutant Flp-EBD yeast library for selection of improved EBD ligand binding. However, it turned out that the vector containing strain exhibit basal Flp activity without ligand binding. Thus, the current yeast strains are not suitable to screen for improved flp activity upon ligand binding due to the basal flp activity. Also Cre shows basal activity in yeast as well and therefore Cre-EBD is also not suitable to improve EBD in this yeast strain. Therefore, we are testing different strains and conditions to overcome this problem beyond funding of this project.
The development of type 3 conditional switches, evaluation of limits of conditional mutagenesis and nuclease technologies
In WP3.3 type 2 switches using Flp and Dre have been successfully implemented in vitro and in vivo. To evaluate potential problems and limitations of conditional mutagenesis, a series of various recombinase expressing vectors and reporter cassettes have been generated, to determine site specific recombinase (SSR) activity as well as toxicity in different species (WP3.4). We could show that SSR toxicity is dependent on SSR activity independent of the location of cryptic sites across the genome. It turned out that there is a hierarchy of SSR toxicity Cre>Dre>Flpo (WP3.4). In the framework of WP3.5 innovative, sequence-specific TALENs and CRISPR guide RNAs against loxP and Frt sites have been constructed. TALENs are fusion proteins of a customized DNA binding domain made of TAL repeats and the FokI nuclease domain whereas CRISPR represents sequence specific guide RNA to which Cas9 nuclease is binding in both cases leading to sequence-specific DNA binding and to site specific double strand breaks within the binding domain. The TALEN/gRNAs has proven highly active in vitro. In addition, an improved standard protocol for the generation of mutants by zygote microinjections of TALEN and CRISPR/Cas9 has been developed and published (Wefers et al. 2013, Schick et al., in revision). Funding for this WP expired after 3 years. Overall the objectives have been achieved, albeit with some adaptations from the original plan by integrating the CRISPR/Cas technology which was not available at the time of application. In addition, this WP includes two lines of exploration that are appropriate to the general goals of technological development for EUCOMMTOOLS but were not defined in the original proposal. These two additional avenues are:
(a) the use of transposition to achieve BAC transgenesis using both PiggyBac and Sleeping Beauty transposons (Bradley and Stewart labs);
(b) the use of designer nucleases to promote targeted mutagenesis and homologous recombination (Skarnes, Kühn/Wurst and Stewart labs).
These extra objectives are based on recent developments in the technical field and were incorporated because they are highly relevant to WP3 goals (Wefers et al. 2013, Schick et al., in revision). Part of this WP will continue after funding.
In summary, WP3 has achieved its goals taking into consideration adaptions due to technical obstacles or novel developments which we integrated smoothly into our research program.
WP4 Embryonic Stem Cell Production
WP Leader: W. Wurst (HMGU), Partners: A. Bradley (WTSI), B. Skarnes (WTSI)
Generation of BAC-CreERT2-T2A-eGFP/Cre-T2A-eGFP transgenic ES cell clones
It turned out that the Cre-ERT2 knock-in strategy taking advantage of EUCOMM intermediate vector resource was the most efficient and stable strategy to establish Cre-drivers at scale. We have established 493 Cre targeted ES cells representing 378 unique genes using this approach. The original proposed BAC-Cre RMCE (recombination mediated cassette exchange) strategy in Hprt or Rosa locus did not result in stable, germline competent ES cell clones by testing 55 different vectors. Therefore, we entirely focused on Cre-knock-in strategy. Based on an efficiency of 60% germline transmission, the injection of 2 clones for each of the 378 targeted genes allowed us to meet our goal of producing 223 strains of Cre knock-in mice (Figure 14). In addition, about 20 vectors are still in production using CRISPR/Cas mediated knock-in in zygotes and 16 in germline testing.
Figure 14. (see attachment)
Thus, we have established sufficient CreERT2 expressing targeted ES cell lines to generate 250 CreERT2 transgenic driver lines.
Production and QC of 3.500 conditional knock-out ES cell lines
In total, in the course of EUCOMMTOOLS 4.501 ES cell electroporations were performed comprising 3.618 genes. All proper annotated genes were processed. We have correctly targeted 2.565 protein coding genes and in addition 392 miRNA genes. In total, 2.957 genes were targeted. Some of the remaining failed genes are not amenable to current mutagenesis technologies. Therefore, we have begun to employ CRISPR assisted mutagenesis using conditional EUCOMMTOOLS targeting vectors. Testing 254 conditional targeting vectors we achieved correct targeting of 146 (58% success rate) genes that previously did not give targeted products. At this point, it can already be concluded that not all genes will be targetable using current conditional strategy and that simple CRISPR/Cas mediated deletion alleles need to be generated for the remaining genes. However, this would also require development and validation of a separate production pipeline.
In summary, we have generated additional 2.957 target genes and have adapted CRIPSR/Cas technology to generate conditional targeted genes by re-using EUCOMMTOOLS targeting vectors.
WP5 Generation of Transgenic Mouse Lines
WP Leader: R. Ramirez-Solis (WTSI). Partner: Y. Herault (ICS), G. Tocchini-Valentini (CNR), N. Rosenthal (Monash/Jax), Steve Murray (Jax), and W. Wurst (HMGU)
Generation of Cre-driver knock-in transgenic mouse lines
The WP5.1 Cre mouse production centers have generated 223 (reflecting 221 genes, 36 lines are still in the pipeline) Cre driver mouse lines by injecting 318 unique Cre-driver ES cells. These lines are crossed to reporter lines (lacZ, tdTomato) to monitor Cre activity in vivo. Especially the last year has seen enormous progress in the production of new Cre-driver knock-in transgenic lines from the all the EUCOMMTOOLS mouse production centres. At the last count just prior to writing this report, 223 transgenic mouse strains have been produced for 221 unique Cre-driver loci. This represents about 88% of our stated goal. A further 16 strains are in the breeding process to achieve germline transmission. The partners ICS, WTSI and CNR have delivered their original requirements (ICS 49/50. WTSI 107/100, and CNR53/50). The two new partners (Monash and Jax) have integrated successfully into the workflow and are raising the total number of lines produced already. These partners currently inject 20 vectors directly into zygotes taking advantage of CRISPR/Cas9 mediated knock-in strategy (which are not included into table 2. The table illustrates the detail of the total number of injections, new strains (# Genes GLT) generated, and strains still in process at each of the participating centers (Table 2).
Table 2. (see attachment)
Table 3. (see attachment)
The next step in the process was to breed the Cre-driver knock-in lines to Cre-reporter strains. When the project started, the chosen reporter line was ROSA26-loxP-STOP-loxP-lacZ. During the development of the project based on SAB suggestion, a decision was made to switch the reporter strain to a tomato fluorescent reporter, namely B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze which provides better cellular resolution. The final proportion of strains on the different reporters is illustrated in the table 3 below. At last count, overall, 58% of the strains were crossed to the lacZ reporter and 42% to the fluorescent tomato reporter.
In addition, SOPs for tamoxifen treatment, perfusion and dissection have been established for CreERT2 transgenic lines together with WP6. Double heterozygote animals (het for the Cre-driver, and het for the reporter locus) were dosed with tamoxifen as follows:
• Adults
• P56 (+/- 3 days) mice were IP injected, once a day for 5 consecutive days and perfused 8 -12 days after the first injection. Each mouse received 100µl (10mg/ml) of tamoxifen solution per day.
• Pups
» P6 (+/- 1 day) mice are dosed with 1 sub-cutaneous injection of tamoxifen solution and perfused at P14 (+/- 1 day). Pups receive 20µl of tamoxifen solution per gram of body weight.
Originally, for each strain 2M and 2F were injected with tamoxifen, including controls. This arrangement was revised at the May 2014 in Rome EUCOMMTOOLS meeting. These are the revised number of samples for each treatment per strain:
» No control pups shipped
» 1 Adult p56 (male) + back up (preferably male, since no gender effects on expression had been observed)
» 2 pups P14 (male + female) + back up (either sex).
Since many mutant mouse strains were made with the drug-resistance cassette still present at the locus in order to avoid the negative effect of the Dre-treatment on the ESC germline capability, four strains were generated with and without Dre-excision of the selectable cassette in comparison. This allowed the evaluation of the effect of the marker on the specificity of expression of the Cre-driver. The Cre-driver loci for the strains Cpne6, Gpx3, Itih1, and Prdm16 loci were chosen. Mutants for both (excised and non-excised) were bred to reporter strains, and double heterozygotes animals were treated as usual and shipped to WP6 for annotation. No differences in Cre-activity have been observed.
Quality control of conditional mutant ES cells
WP5.2 served as quality control of produced ES cells in respect to germline transmission efficiency. In contrast to the originally proposed 50 transgenic mouse lines, more than 5.000 mutant mice have been established using EUCOMM/EUCOMMTOOLS ES cells generated by WP4.2 as well as by EUCOMM/KOMP. The mouse production was mostly performed by IMPC members. Thus, quality control of the ES cell resource has been provided extensively. All transgenic mice established in the framework of EUCOMM/EUCOMMTOOLS are archived for distribution by EMMA/Infrafrontier (www.infrafrontier.eu). Some of the Cre-driver lines will be in addition available via the Jackson Laboratory (www.jax.org).
In summary, the establishment of Cre-driver lines and conditional target mouse strains has been achieved.
WP6 Cre Expression Annotation
WP leader: S. Martinez (UM)
WP6 has developed a reference atlas of anatomical structures at P7, P14 and P56 mouse to annotate Cre expression at the cellular level. An ontogenic annotation trees have been developed for standardized nomenclature. We have established adequate and reproducible conditions to get perfect high quality sections for histological analysis. The whole bodies of P14 mice have been processed in sagittal and transversal sections. Twenty sagittal sections have been processed in different series to detect lacZ or tdTomato expression and GFP by immunostaining in combination with specific cell markers. All the body organs are represented in sagittal sections, which gave us an overview of the general expression pattern. These expression patterns have been compared with publicly available expression data bases (Eurexpress, Genepaint, Allen brain project).We have generated an expression maps in relation to the reference atlas. According to the suggestions of the SAB WP6 focused on the analysis of 1 male, 1 female at P14 and 1 male at P56 offsprings to enhance annotation efficiency. At present, tissues of 193 Cre-driver stains have been obtained. Among them, 74 Cre-driver lines expression annotations have been achieved: Tns1, Krt17, Acan, Mb, Tshb, Prdm16, Cpne, Ucp1, Gpx3, Heatr8, Tle6, Trpv1, Smyd3, Hoxa2, Tac2, Pou2f1, Rbpjl, Spp2, ROSAL2/WT, Ptprcap, Cd3e, Atp6v1g2, Ccl25, Hes5, Myo1a, Insl3,Csk/Csktm1, Dmbx1, Mia, Spink8, Entp5, Acsm4, Dbx1, Cdx2, Gnai2, Pkd2l1, Sfrp1, Cnp, P2rx7, Pdgfrb, Gast, Cd63, Cpne6, Nefh, Amhr2, Rbfox3, Wnt9a, Barhl1, Zfp819, cldn7, Sgca, Pbx2, Zbtb32, A830010M20F, Lztfl1, Ttl25, Sds, Aqp3, Eomes, Spic, Zfp629, Slc26a5, Fga, Tbx2, Tff1, Cmtm5, Ttr, Myh4, aldh3a2, Igj, Slc26a4, Mog, Cdh2, and Sost.
A total number of 85.577 annotations at the body and brain have been performed resulting in expression annotation to 15.082 cell types.
All brain and body annotations together with selected images are published on our online platform at www.imib.es/AnotadorWeb. The following images show representative examples of Cre-driver (Tomato) lines annotated from body (Figure 15) and brain sections (Figures 16, 17) representing the expression of the endogenous genes to almost 100%.
Figure 15. (see attachment)
Figure 16. (A, B) (see attachment)
Moreover, the analysis of the expression of the new reporter line based on a fluorescent protein reporter (Tomato) clearly shows an advantage for the annotation of specific type of cells in the brain (Figure 17).
Figure 17. (see attachment)
Taken together, 74 lines have been annotated. Thus, we are behind the original plan date because of the delay in CreERT2 ES cell provision based on changing strategies from BAC- CreERT2 to Knock-in CreERT2 lines
WP7 Phenotyping of Cre Transgenic Mice
WP Leader: S. Brown (MRC)
In WP7 BAC-CreERT2 transgenic lines were planned to be phenotyped to determine potential by-stander effects of additional genes located on the BAC. Since we changed strategy towards knock-in into the endogenous locus and pre-selected promoters/genes showing no haploinsufficiency when mutated we did not expect phenotypic alterations in these transgenic mice. Nevertheless, eight Cre-ERT2 transgenic knock-in lines have been selected and six lines were imported by partner 8 and have been assessed for phenotypic consequences of Cre expression and of potential haploinsufficiencies. To date, phenotyping of lines Gpx3, Ucp, Mb and Tshb have been completed. As expected, due to the change of strategy no phenotypic alterations were found after intensive primary screening in the Harwell mouse clinic (see also IMPC phenotyping pipeline) compared to wildtype littermates. This work is performed exclusively by institutional funds of MRC Harwell.
Taken together, phenotyping of Cre-driver lines did not result in phenotypic alterations.
WP8 Bioinformatics
WP Leader: B. Skarnes (WTSI), Partners: S. Martinez (UMH), W. Wurst (HMGU)
WP8 (Bioinformatics) was supporting the design, tracking and reporting of eGFP-CreERT2 vectors, of the artificial intron containing conditional targeting vectors of EUCOMMTOOLS, of CRISPR/Cas mediated targeting as well as data tracking within the consortium. In addition, it is integrating data and maintaining the EUCOMMTOOLS/IKMC/IMPC web portal. The production of Cre knockin ES cells based on EUCOMM intermediate vectors has been tracked in our main production LIMS and the production of Cre knockins in mice is tracked in iMITS. All of the information on Cre knockin ES cells as well as mouse production have been uploaded into iMITS. iMITS has been migrated to the EBI, Hinxton where the data is archived and displayed on the IMPC website (www.mousephenotype.org). The informatics activities of the IKMC - which includes EUCOMMTOOLS - have now been transferred to the IMPC for long term sustainability reasons. The project website can always be accessed via http://www.eucommtools.org which continues to direct the user to the EUCOMMTOOLS project website, now embedded into IMPC website. WP8 has achieved its goals by supplying all actual bioinformatics support needed by the production work packages.
WP9 International Coordination and Databases
WP Leader: N. Rosenthal (MONASH), Partners: A. Bradley (WTSI), W. Wurst (HMGU)
The goals of the EUCOMMTOOLS International Coordination Committee (ICC) were to promote international collaboration and the adoption of common standards and committed to the project. We included stakeholders from around the world, coordinated activities between the existing EU coordination action CREATE (dev.creline.org) and the major non-European Institutions building similar resources, including NIH (US), NINDS (US), Scripps Institute (US), JAX/IMSR (US), Allen Institute (US), RIKEN (Japan), MSHRI (Canada) and APN (Australia). The aim of WP9 was to align activities between centres, to assimilate their recommendations and reports, and to continue developing a technology roadmap for mouse conditional mutagenesis and generation of human disease models.
Members of the ICC (and corresponding Cre driver related initiatives) have been:
• Nadia Rosenthal (Monash University, Australia, CREATE consortium coordinator)
• Colin Fletcher (NIHGRI, USA) (KOMP, Cre Driver Network)
• Janan Eppig, Steve Murray (The Jackson Laboratory, USA)
• Colin McKerlie, Lauryl Nutter, Marina Gertsenstein (TCP, Canada) (NorCOMM2ls)
• Elizabeth Simpson (UBC, Canada) (CanEuCre)
• Geoff Hicks (U Manitoba, Canada) (NorCOMM, NorCOMM2ls)
• Andras Nagy (MSHRI, Canada) (NorCOMM2ls, Cre-X-Mice)
• Hongkui Zeng (AIBS, USA) (AIBS Transgenic Programme)
• Kent Lloyd (UC Davis, USA) (KOMP, MMRRC)
• Michael Dobbie (Australian Phenomics Facility - APF, Australia)
• Nat Heintz, Laura Kus (Rockefeller U, USA) (GENSAT)
• Yuichi Obata, Atsushi Yoshiki ((RIKEN-BRC, Japan) (RBRC)
WP9 partners organized meetings in the framework of EUCOMMTOOLS, IKMC and IMPC meetings (July 5-7, 2011: EUCOMMTOOLS Progress Meeting (Rome, Italy); December 2-4, 2013: INFRAFRONTIER-IMPC-IKMC Meeting and EUCOMMTOOLS workshop (Rome, Italy); October 26-30, 2014: MGI 25th Anniversary Celebration at The Jackson Laboratory (Bar Harbor, USA)); as well as ICC conference calls agreeing on common nomenclature and SOPs for generating and annotating Cre drivers, implement web services in order to link the respective databases, disseminate conclusions and SOPs, develop plans for long term sustainability and distribution of the resource as well as emerging technologies for Cre driver generation relating to gene editing.
WP 10 Distribution and Archiving
WP Leader: A. Bürger (HMGU)
The WP10 objective is to import, expand, quality control, archive and distribute all EUCOMMTOOLS vectors and mutant ES cells via the EuMMCR (European Mutant Mouse Cell Repository). EuMMCR was founded as the EUCOMM Material Distribution Unit, which archives, distributes, and quality controls EUCOMM and EUCOMMTOOLS materials, i.e. conditional gene trap and conditional gene targeting vectors as well as EUCOMM gene targeted and gene trapped ES cells. Distribution is performed internally (to EUCOMM and EUCOMMTOOLS mouse production centers) and externally, to the scientific community worldwide. EuMMCR has established its own website (www.eummcr.org) which informs the user about the available resources and the ordering procedures and ensures a better and easier access to and visibility of the projects’ resources.
ES cell distribution: Upon orders from interested scientists worldwide, the EuMMCR lab is expanding requested ES cell clones and refreezing those clones in several aliquots, which allows EuMMCR shipping aliquots to the requesting scientists and maintaining the resource. Furthermore, the EuMMCR lab is performing quality controls on the ES cell samples like e.g. mycoplasma test, verification of the identity of the mutated allele (PCR), development of a genotyping protocol (PCR), verification of the correct allelic integration by long range PCR (LRPCR) over the 5’ and 3’ homology arms of the targeting vector, and counting the copy number of neo and lacZ integration (by qPCR) to exclude double or multiple integrations of the targeting cassette.
Import of EUCOMMTOOLS resources: More than 16.500 conditional EUCOMM/EUCOMMTOOLS vectors were imported from partner 2. The vectors have been delivered in 96well and 384well plates as glycerol stocks. Upon arrival the vector plates were immediately stored at -80°C. The EUCOMM and EUCOMMTOOLS ES cell clone collections in the EuMMCR storage facility is currently composed of approximately in total 2.3 Mio samples.
EuMMCR production and distribution numbers: EuMMCR production started in 2008. The production and distribution numbers from 2008 until September 2015 (expected numbers until end of 2015 are shown in brackets) are shown in Figure 18. EuMMCR has distributed a total of 11.903 (12.098) ES cell clones to the international scientific community for transgenic mouse production. Of these, 9.441 (9.636) ES cell clones were delivered since the beginning of EUCOMMTOOLS. The differences in distribution to production numbers are due to quality control failures, which are not sent out to the end user.
Vector distribution started in 2009 and has now reached a total of 926 (959) vectors as shown in Figure 19. Overall the number of requests (not shown) and distributions show that the EUCOMM and EUCOMMTOOLS resources are well received in the scientific community worldwide.
Figure 18. (see attachment)
Figure 19. (see attachment)
mirKO resource: The Sanger Institute’s mirKO resource is a collection of targeting vectors and heterozygous targeted miRNA genes in JM8.A3 ES cells using a gene deletion strategy. In total, 212 targeted alleles (clusters and singleton knockouts) corresponding to 253 different miRNA genes have been sent to EuMMCR for archiving and stored in liquid nitrogen storage and are ready for distribution upon requests. Each microRNA gene is represented in the archive with up to 6 individual ES cell clones. The resource was published (Prosser HM et al. Nat Biotechnol. 2011; 29(9):840-5.).
Cre-ERT2 Knockin ES-Cell resource: A large collection of 375 recombinant ES cells have been established and archived. The EuMMCR imported this very important resource from EUCOMMTOOLS partner 2 (WP 4.1) into our archive, as well as the corresponding data. Thus, Cre-ERT2 vector and cell resource is also available to the scientific community. Up to now, we imported more than 28.000 samples from these Cre-Driver lines.
Strategic partnership with genOway: EuMMCR recently entered a strategic partnership with genOway. This allows us to distribute all products available at EuMMCR to “for-profit” organizations. Industry scientists benefit from stringent genOway quality controls and the unmatched reliability of its platform in the production of the EUCOMM and EUCOMMTOOLS conditional KO models for commercial partners. For more information please refer to http://www.genoway.com/products-services/eucomm/eucomm-conditional-knockouts.htm
Ordering process: The ordering process has been re-established in 2013. Now, it is possible for external requestors to search ES cells and vectors on the EuMMCR homepage (http://www.eummcr.org) and order them directly from the website. After finishing the order form, requestors receive an automatically created MTA in the PDF format by email. In order to finalize the order, the requestor prints the MTA, signs it and sends it to the legal department of the Helmholtz Zentrum München (HMGU).
Quality control: An important issue for WP10 is the quality control of the ES cell clones. Since January 2014, all the following quality controls are applied to every clone that the EuMMCR sends out. This is a huge improvement of the ES cell quality.
a. Mycoplasma PCR: all ES cells expanded and shipped by EuMMCR are tested for mycoplasma contamination using a PCR mycoplasma assay.
b. loxP PCR: the conditionality of the allele directly depends on the presence of both loxP sites flanking the critical exon. Due to recombination events within the area of the targeted region a loxP site can be absent in some of the EUCOMM alleles. The results of such recombination events are clones reflecting null mutation “without conditional potential”. Therefore EuMMCR is verifying the presence of both loxP sites, called “loxP PCR”, for all alleles.
c. genotyping: EuMMCR develops a genotyping protocol allowing the end user to genotype the ES cells and mutant mice easily. This genotyping LRPCR is performed with a primer in the targeting vector of the EUCOMMTOOLS allele and two gene specific primers outside the vector to confirm homologous recombination.
d. qPCR, copy number and chromosome check: In order to enable to exclude ES cell clones with double or multiple integrations of the targeting cassette, EuMMCR has established a neo and lacZ count qPCR. In addition, we check the chromosome numbers of Y, 8 and 11 to quickly exclude chromosomal alterations like trisomy 8 or 11, or loss of the Y chromosome.
WP 11 Training, Sustainability, Knowledge Dissemination and Outreach
WP Leader: W. Wurst (HMGU); Partners: all teams from WTSI, EMBL, TUD, CNR, GIE-CERBM, UMH and MRC
WP11 is concerned with the project’s training, sustainability, knowledge dissemination and outreach issues. To stringently manage and organize the EUCOMMTOOLS novel developments and production of conditional mutant materials monthly telephone conferences, meetings, and exchange with SAB have been organized. Face to face meetings of the scientists, steering committee and SAB took place regularly.
Monthly telephone conferences:
EUCOMMTOOLS organized regular internal consortium telephone conferences during the whole duration of the project to discuss project progress and problems. In general, these telephone conferences took place about once a month, with the exception of months where a progress meeting is organized, and of August (summer break). In addition, 10 consortium meetings took place to discuss progress, limitation, strategies, and novel developments to be integrated into the program. These meetings were also used for meetings of the steering committee and the scientific advisory board.
Consortium meetings:
- October 4-5, 2010: EUCOMMTOOLS kick-off meeting (Unterschleißheim, Germany)
- April 5-6, 2011: EUCOMMTOOLS Progress Meeting (Alicante, Spain)
- July 5-7, 2011: EUCOMMTOOLS Progress Meeting (Rome, Italy)
- April 3-4, 2012: EUCOMMTOOLS Progress Meeting (Hohenkammer, Germany)
- December 18-19, 2012: EUCOMMTOOLS Progress Meeting (Unterschleißheim, Germany)
- June 10-11, 2013: EUCOMMTOOLS Progress Meeting (Hinxton, UK)
- December 2-4, 2013: INFRAFRONTIER-IMPC-IKMC Meeting and EUCOMMTOOLS workshop (Rome, Italy)
- May 27-28, 2014: EUCOMMTOOLS Progress Meeting (Unterschleißheim, Germany)
- June 19, 2015: EUCOMMTOOLS Airport Meeting (Munich, Germany)
- September 3-4, 2015: EUCOMMTOOLS Farewell Meeting (Hohenkammer, Germany)
Steering Committee Meetings:
Meetings of the EUCOMMTOOLS Steering Committee (SC) took place in the framework of each of the EUCOMMTOOLS meetings mentioned above.
Scientific Advisory Board Meetings:
The EUCOMMTOOLS Scientific Advisory Board (SAB) consists of:
• Janan Eppig, The Jackson Laboratory, Bar Harbor, ME, USA
• John Hohmann, Allen Institute for Brain Science, Seattle, WA, USA (2011-2014)
• Alexandra Joyner, Memorial Sloan Kettering Cancer Center, New York, NY, USA
• Monica Justice, Houston, The Hospital for Sick Children (SickKids) in Toronto, Canada
• Björn Löwenadler, AstraZeneca R&D, Mölndal, Sweden
• Andrew McMahon, Harvard University, Cambridge, MA, USA
• Luis Puelles, University of Murcia, Spain
Three (J.E. B.L. and A.M.) of these advisors have already been members of the EUCOMM Scientific Advisory Board, so that they support continuity between the two projects.
The EUCOMMTOOLS SAB met once a year to give scientific advice to the project. According to this planning, the SAB was invited to the EUCOMMTOOLS progress meeting in Alicante, Spain (2011), the EUCOMMTOOLS progress meeting in Hohenkammer, Germany (2012), the EUCOMMTOOLS progress meeting in Hinxton, UK (2013), the EUCOMMTOOLS progress meeting in Unterschleißheim, Germany (2014), and the EUCOMMTOOLS farewell meeting in Hohenkammer, Germany (2015). Within these meetings the SAB discussed the project progress and developments with the EUCOMMTOOLS SC in separate sub-meetings and gave advice throughout the project.
Training courses:
Several training activities of ‘The Bioinformatics of Mutant Mouse Resources’ were performed by the EUCOMMTOOLS members EBI, WTSI and MRC Harwell. These training activities have been offered at MRC Harwell, UK on December 6-7, 2010 and, in addition, as a satellite workshop at the Mouse Genetics Meeting in Washington, DC, USA (June 22-25, 2011). The objectives of these two-day hands-on activities were, among others, to show participants how to find critical information about their favourite genes and to help them understand gene structures in detail as well as the underlying experimental data supporting the annotation. In addition, participants were enabled to find mutant mouse resources for their genes of interest using the IKMC and similar resources, to search IMPC phenotypic databases such as EuroPhenome and to gain an understanding of the high-throughput phenotype data generated and the statistical approaches required to analyse it.
Furthermore, a one day condensed version of the training was offered before the start of the Mouse Molecular Genetics Conference in Hinxton, UK (September 20, 2011) and was followed up with an afternoon session on mouse mutant resources during the 17:00-19:00 poster session on Sep. 21, 2011.
Responses to these training courses were very positive, and although originally not planned in Annex I, this training activity has continued during the reporting periods 2 and 3, having been offered at MRC Harwell, UK (June 18-19, 2012) and the European Bioinformatics Institute (EBI), UK (July 11, 2012), at Salamanca as part of a half-day workshop for the International Mammalian Genome Conference, Spain (September 15, 2013) and Hinxton, UK (September 19, 2013).
The advanced course “Genetic Manipulation of ES Cells” took place twice at the Wellcome Trust Sanger Institute Genome Campus in Hinxton, UK (October 31 - November 13, 2011; November 5 - November 18, 2012). This event was a two week intense practical and lecture course on gene targeting and genetic manipulation of mammalian stem cells, emphasizing mouse ES cells but including human iPS cells as well. Topics comprised gene annotation and KO design, vector construction through recombineering, ES cell culture, gene targeting, transposon technology, site-specific recombinases and RMCE, modular IKMC vector resources, genotyping and mouse production, homozygous ES cells, and TALEN assisted gene targeting. This course was originally not planned in Annex I but performed in addition.
The advanced course “Genetic Engineering of Mammalian Stem Cells”, which also took place at the Wellcome Trust Sanger Institute Genome Campus in Hinxton, UK (March 10th -22th, 2014), was a two week intense laboratory-based training course providing a comprehensive overview and practical laboratory experience of the genetic manipulation of mammalian stem cells. Focusing on mouse ES and human iPS cells, recent advances in genome informatics, recombineering, custom nucleases (CRISPRs/TALENs), transposon technology and conditional gene targeting were covered in lectures and through interactive demonstrations. Laboratory work included the culture and transfection of ES cells, design and construction of gene targeting vectors by BAC recombineering, and the use of PiggyBac transposons and designer nucleases (CRISPRs/TALENs) for advanced genome engineering. Participants were trained in the informatics and practical use of public gene targeting resources, including the production of mutant mice from the IKMC (International Knockout Mouse Consortium) resource. Emphasis was placed on the planning/design and successful experimental execution of gene targeting projects.
Two workshops were organized by Salvador Martinez, Almudena Martinez Ferre and Marusa Arencibia and took place at UMH Alicante, Spain. The workshop “Mouse Perfusion and Dissection” (May 7, 2013) aimed to instruct EUCOMMTOOLS work package 5.1 members in mouse perfusion and dissection, so that they are able to prepare samples for work package 6. The course was attended by Francesco Chiani and Alessia Gambadoro (CNR), Bruno Weber (ICS) and Ellen Brown, Nicola Griggs, Caroline Sinclair and Elizabeth Tuck (Sanger). Salvador Martinez (EUCOMMTOOLS work package 6 leader) introduced the course by describing the methodology and procedure for perfusion of P14 and P56 mice and visceral block extraction. Participants got to know the best fixation protocol for EUCOMMTOOLS samples, all suitable materials to be used and how to process the samples properly by immunohistochemistry. During the practical training, Salvador Martinez and Almudena Martinez showed how to perfuse P14 and P56 mice in situ and how to dissect the brain as well as thoracic and abdominal blocks of P56 mice. The second workshop “Tutorial on Mouse Anatomy and Gene Expression Patterns” (September 28-29, 2015) was designed to present the members the process of genetic annotation in relation to the project. The first part of the course had a theoretical approach in which knowledge were provided on the comparative anatomy of mouse and human body and the brain, being based on the ontology elaborated for the project. In the second part, the participants to the course applied all knowledge acquired in the theoretical sessions. Participants used the annotation platform that was designed for annotation. In particular, participants learned to use the annotation webpage and each of them annotated the expression of the gene reporter (Cre) of two samples of the project for both, the body and the brain. Moreover, they carried out a comparative expression analysis with other data bases. This was a very dynamic and interactive course and all participants acquired new knowledge on histology, and gene expression annotation.
Potential Impact:
Part IV: EUCOMMTOOLS Potential Impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results
Strategic impact
To be able to fight a disease efficiently, its cause(s) must be known. In particular, to understand the genetic contribution to disease etiology, genetic modified animal models are mandatory. These models are an absolute requirement to elucidate the function of (newly) identified disease genes, since knowing the function of a gene is essential for understanding the related disease processes. The physiological function of a gene can best be described via its loss-of-function in the whole organism. Thus the overall vision of the EUCOMM/EUCOMMTOOLS programs was to build the basis to the annotation of the function of all protein coding genes by producing mouse mutant resources for the world wide scientific community as basis to generate mouse models for elucidating gene functions.
In this regard EUCOMM/EUCOMMTOOLS are by now one of the key providers of mouse mutant resources worldwide and was a founding members of International Knockout Mouse Consortium (IKMC, http://www.mousephenotype.org) which provides the fundament of the International Mouse Phenotyping Consortium (IMPC, http://www.mousephenotype.org). Thus, together, in these international consortia null mutations of all protein coding genes are generated and their primary phenotypes are determined and an encyclopedia of gene function is currently generated. This encyclopedia will also form the reference for further more sophisticated disease models in which disease associated alterations will be recapitulated in vivo (i.e. introduction of disease associated mutations). All mutant embryonic stem (ES) cell and vector resources produced by EUCOMM/EUCOMMTOOLS are archived by the European Mouse Mutant Cell Repository (EuMMCR, http://www.eummcr.org) and all mutant mice at the European Mouse Mutant Archive (EMMA, http://www.infrafrontier.eu).
The success of EUCOMM/EUCOMMTOOLS programs is reflected by the fact that already more than 18.000 mutant ES cell have been produced by gene targeting and gene trapping by IKMC of which about 13.000 have been contributed by the EUCOMM/EUCOMMTOOLS consortia. It has to be noted that this responds to a coverage of the mouse genome of about 90%. The remaining 10% were not amenable to the EUCOMMTOOLS technology but can now be tracked by CRISPR/Cas9 technology. Proof of concept for large scale production has also been provided by EUCOMMTOOLS. More than 12.000 mutant ES cell clones and 1.000 gene targeting vectors have been distributed by EuMMCR to the scientific community upon request. Furthermore, based on this IKMC resource more than 5.000 mutant mice have been produced mostly by members of the IMPC and are currently phenotyped by IMPC and Infrafrontier and results are provided to the public. In addition, about 1.770 EUCOMM/EUCOMMTOOLS mouse lines have been archived by the EMMA project, and already about 1.489 of these mouse lines have been distributed to the international scientific community. The distribution of EUCOMM/EUCOMMTOOLS mice constitutes about 50% of the total EMMA/Infrafrontier distribution activities (pers. comm. Dr. Michael Hagn, EMMA project manager).
Concerning disease models the fact that for over 1.320 OMIM (Online Mendelian Inheritance in Man)-defined human genetic diseases at least one experimentally defined mouse model is available encompassing over 4.450 mouse models most of which are derived from IKMC, ECOMM/EUCOMMTOOLS mutant resource material (http://www.mousephenotype.org http://www.diseasemodels.org) (Eppig et al., NAR, 2015, de Angelis HM et al., 2015) also demonstrates the success of these programs and their absolute necessity for the scientific and medical community. In addition over the recent years it became apparent that many diseases present themselves as “syndromes”, that is, they present themselves with comorbide phenotypes. Thus the elucidation of gene function in specific organs and cell types, understanding the pleiotropic function of genes, is also crucial to understand disease etiology. To address this question conditional mutants are required which are provided by EUCOMM/EUCOMMTOOLS by the versatility of its products (“knock-out first, conditional ready”) since the loss-of-function mutant mice generated in the first instance can be simply converted to conditional mouse mutants by a single cross with another mouse line (i.e. the Cre-driver mouse line). Using these conditional mouse lines a gene can be inactivated in specific organs and cell types as well at specific time points by crossing them to appropriate Cre-driver lines, expressing a Cre-recombinase capable of inactivating the conditionally mutated gene in specific cell types and at specific time points during the lifespan of the mutant mouse, respectively.
In summary, the EUCOMM/EUCOMMTOOLS resources are heavily used by the scientific community as demonstrated by the distribution numbers of EuMMCR and EMMA/Infrafrontier and has substantially advanced the in vivo analysis of gene functions by IMPC/Infrafrontier and thus of the molecular genetic mechanisms underlying hereditary diseases (http://www.mousephenotype.org). Moreover, more than 1000 publications so far have acknowledged the use of EUCOMM/EUCOMMTOOLS material (google scholar) reflecting furthermore the success of these resources. Furthermore, the biotech company genOway is now distributing EUCOMM/EUCOMMTOOLS material to the pharmaceutical industry (see press release http://www.genoway.com/products-services/eucomm/eucomm-conditional-knockouts.htm). Thus, EUCOMM/EUCOMMTOOLS products are also of commercial value and have impact on European competitiveness in biotech and pharmaceutical industry.
European approach
The knowledge, technological expertise and experience as well as infrastructure brought together for the EUCOMMTOOLS proposal can certainly not be found in a single institution or country; therefore, this consortium has been built on a Pan-European level. In addition, no single country could afford the finances for a project of this dimension, thus, EU funding has been necessary to implement this undertaking. Europe has already taken the lead in a number of new research approaches that are fundamental for the future use of the mouse as a central model organism (e.g. the EU programmes EUCOMM/EUCOMMTOOLS, I-DCC, CREATE, EUMODIC, Infrafrontier). Among these projects, certainly, the EUCOMM/EUCOMMTOOLS program laid the groundwork for Europe’s leadership in high-throughput gene function annotation. The mission of the EUCOMM/EUCOMMTOOLS project has been, as detailed below, to provide sophisticated tools enabling researchers world-wide to capitalize on the EUCOMM/EUCOMMTOOLS and IKMC resources, as these resources will allow an unprecedented fine-tuning of disease analysis for about 90% of all protein coding genes. Thus, EUCOMMTOOLS was instrumental in consolidating and even extending Europe’s leadership in large scale and mouse functional genomics. In particular, it also lays the ground, know-how and tools to functionally validate the outcome gathered in systems biology research approaches in vivo. In addition, the EUCOMMTOOLS vector toolkit allows introducing various human disease associated alleles for mimic human diseases, thus, EUCOMMTOOLS will build a bridge between research using cutting-edge mouse models and approaches using human disease associated alleles. Furthermore, all vector tools produced by EUCOMMTOOLS can readily be used to study disease mechanisms in human ES and iPS cells.
The EUCOMMTOOLS project is highly integrated with other national, European and international initiatives:
1) In the EUCOMMTOOLS work packages knowledge and results generated by several other EU programs are directly included, as the CREATE program in work packages 1 and 9, EURExpress in work package 1 and, as the Cre expression analysis is done by an expert also involved in EURExpress gene expression annotation, also in work package 6, as well as EUMODIC in work package 7, as the EUMODIC coordinator will perform mouse phenotyping in this work package according to the EUMODIC standards. In addition, a national United Kingdom initiative will be integrated into EUCOMMTOOLS, namely the establishment of the knockout miRNA resource “mirKO” at the Wellcome Trust Sanger Institute which is supported by institutional Wellcome Trust funds.
2) The I-DCC consortium has been a close EUCOMMTOOLS collaboration partner and integrated all mouse genomics and functional genomics data in one IKMC web portal (http://www.knockoutmouse.org). Furthermore, the EUCOMMTOOLS consortium tightly cooperates with the EMMA/Infrafrontier consortium, which distributes the Cre driver and conditional knockout mice produced by EUCOMMTOOLS and the scientific community. On the international level, EUCOMM/EUCOMMTOOLS contributed certainly most resources to IKMC, which is composed in addition of KOMP, NorCOMM, and TIGM. In addition, a NorCOMM successor proposal (NorCOMM2) which is generating Cre driver lines mostly expressed in stem cell populations using a transposon approach (coordinator: Colin McKerlie). This undertaking is complementing the EUCOMMTOOLS approach. EUCOMMTOOLS tightly cooperates with NorCOMM2 to prevent redundancies. Furthermore, the group of Prof. Simpson at the University of British Columbia coordinated the CanEuCre proposal in close cooperation with EUCOMMTOOLS. The goals of this proposal were to establish 30 Cre driver transgenic lines using predetermined mini and maxi promoters and a knock-in approach in the Hprt locus. Furthermore, 25 AAV virus containing Cre vectors have been generated for direct application in vivo. CanEuCre is integrated into, and its materials registered and distributed by, the IKMC. CanEuCre was a close collaboration partner of EUCOMMTOOLS, also complementing its Cre driver approach.
3) Via its work package 9 ‘International coordination and databases’ EUCOMMTOOLS is integrated into the IKMC/IMPC database. This WP also coordinated international effort to harmonize SOP, to prevent overlaps and redundancy to generate synergies to create a unique resource under the umbrella of IKMC and IMPC.
In summary, the EUCOMM/EUCOMMTOOLS program is an extremely well integrated initiative which is part of a singular international undertaking representing the world’s largest collection of, predominantly conditional, mutant murine materials (vectors, vector toolkits, ES cells, Cre driver and knockout mouse models), easily searchable via the common IKMC web page (www.mousephenotype.org) which is connected to the various IKMC members’ databases and which material is readily available and distributed via EuMMCR, MMCR and EMMA/Infrafrontier to academic and commercial customers worldwide.
Dissemination of Project Results
Dissemination of Knowledge
In general, the scientific results of EUCOMMTOOLS have been disseminated to the scientific community by the classical means of publication in peer review scientific journals and by presentation at scientific conferences and webportals. So far, 167 manuscripts of EUCOMMTOOLS members have been published related to the EUCOMMTOOLS project. Moreover, already more than 1000 publications acknowledged the use of EUCOMM/EUCOMMTOOLS material (google scholar, December 2015).
In addition, we introduced the EUCOMMTOOLS project to the scientific community by producing flyers and posters. Furthermore, as known from the EUCOMM program, a resource producing project’s home page has been established (http://www.eucommtools.org which directs the user to the EUCOMMTOOLS project website, now embedded into IMPC website www.mousephenotype.org ). As EUCOMMTOOLS builds on and enhances the EUCOMM resource, intentionally the EUCOMMTOOLS name and logo have received the characteristics of a EUCOMM name and logo add-on, and a common EUCOMM/EUCOMMTOOLS/IKMC/IMPC home page was established which is now being integrated into the IMPC web portal. The latter is particularly important as EUCOMM is already well-known, as also stated by the EUCOMM reviewers, in the wider scientific community. Thus, EUCOMM has acquired ‘brand’ characteristics which should be extendable to EUCOMMTOOLS via public relations measures. In addition, the EUCOMM/EUCOMMTOOLS home page is approachable via the IKMC home page, and EUCOMMTOOLS’ data integrated into the IKMC searchable database. On the EUCOMM/EUCOMMTOOLS home page also all protocols for handling of the consortia’s materials will be found, e.g. for electroporation, cell culture, microinjection etc.
EUCOMMTOOLS’ knowledge will also be disseminated via the other EU projects directly integrated into the consortium and by networking with different national, European, and international projects, as e.g. several projects funded within the eMed Systems Medicine Research Network, Infrafrontier, and the International Mouse Phenotyping Consortium (IMPC).
EUCOMMTOOLS’ work package ‘International coordination and databases’ also contributes substantially to international collaboration in collating information on the activities in Europe and worldwide in the field of conditional mouse mutant technologies, determining the degree of integration and discussing ways to improve coordination. All international activities have been coordinated with our IKMC partners.
Dissemination of EUCOMMTOOLS materials
In addition to knowledge, the materials produced by EUCOMMTOOLS are disseminated to the scientific community by the EuMMCR (vectors, toolkits, ES cells) and by the EMMA/Infrafrontier consortium (mice).
The EuMMCR (http://www.eummcr.org) evolved in the framework of the EUCOMM project as EUCOMM’s material distribution unit, but also archives and distributes material of other EU funded projects such as EuTRACC and SYBOSS materials. The EuMMCR became part of the Helmholtz Zentrum München in November 2007 and, since then, predominantly distributes quality controlled mutant ES cells (more than 12.000 clones to date), but also gene targeting (more than 1000 to date) and gene trapping vectors to the academic scientific community world-wide. EUCOMM/EUCOMMTOOLS material is now also distributed to commercial partners via genOway worldwide (see press release at the EuMMCR homepage, www.eummcr.org). EuMMCR’s customers are predominantly located in Europe (51%) and the US/Canada (40%), and 9% in other countries. In addition, the EuMMCR also expanded, quality controlled and shipped another about 2.500 clones to IMPC members for mouse production. The EuMMCR is a very successful distribution unit with proven qualification to distribute EUCOMMTOOLS vectors, toolkits and mutant ES cells under quality controlled SOPs efficiently (see also work package 10).
The distribution of EUCOMM/EUCOMMTOOLS mice is performed by EMMA/Infrafrontier. So far, more than 2.000 EUCOMM/EUCOMMTOOLS derived mouse strains have been archived, more than 1.700 strains have been ordered and more than 1.500 have been distributed. In addition, mice produced by The Jackson Laboratory will be distributed by this institution. All EUCOMM and EUCOMMTOOLS materials are/will be distributed to the academic and industrial scientific, and thereby biomedical, community. Even after the end of the funding of EUCOMM/EUCOMMTOOLS all material will be maintained and distributed to the scientific community by a long term self-sustaining concept providing the material to the customer at cost prize. Thus, EUCOMM/EUCOMMTOOLS has established a long term self-sustainable concept far beyond direct funding of the EU.
Commercial exploitation of project results, and plan for management of knowledge (Intellectual Property)
Commercial exploitation issues are taken care of by the Wellcome Trust Technology Transfer Division (WTTTD) together with Ascenion GmbH as counselors of the management team and the steering committee as well as by the Technology Transfer Units of all EUCOMMTOOLS participants. These units have settled a deal with genOway to distribute EUCOMM/EUCOMMTOOLS material to commercial end users. The EUCOMMTOOLS conditional knockout resources are now also available to commercial entities.
In principle, all EUCOMMTOOLS products are of commercial interest, i.e. gene targeting vectors, gene targeting vector design software, conditionally targeted mouse ES cells, conditionally targeted mice, Cre driver mice, as well as products of the EUCOMMTOOLS technology development work package, namely recombinases, new gene cassettes, new recombinase inducible systems etc. For example, conditionally mutated ES cells, and mice derived from such ES cells, are of interest for companies performing pharmaceutical research and development. Besides the elucidation of gene functions, ES cell clones can be used by biotech and pharma companies for preclinical research and development, typically in the so-called ADME&Tox applications (evaluation of the adsorption, distribution, metabolism, excretion and toxicology profile of a compound of interest). Furthermore, these models are of extreme value for drug target validation to determine potential side effects. Moreover, we seek intellectual property protection whenever possible supported by WTTTD and Ascenion GmbH.
As stated above, we have established a commercial distribution via the SME genOway (France). For more detailed information please follow the link to the press release “genOway offers industry scientists immediate access to EUCOMM conditional KO mouse models” which can be found at: https://www.eummcr.org/ .
EUCOMMTOOLS distributes its resources to as many for-profit entities as possible to maximize the commercial as well as the scientific impact of the resources. EUCOMMTOOLS does not entertain exclusive arrangements with any commercial entity for access to the project’s technology developments or resources. In general, the EuMMCR distributes vectors and ES cells to industry via genOway, and mice will be distributed to industry by EMMA/Infrafrontier or the consortium participant who has generated the mice via genOway as well. The revenues for the commercially distributed materials have to be divided between the EUCOMMTOOLS members. In addition, the EuMMCR, when involved, and the technology transfer unit shall receive remuneration. An adequate split scheme is defined in the project’s consortium agreement.
Finally, the commercial exploitation of EUCOMMTOOLS materials has also got high socio-economic impact as e.g. the use of mutant ES cells and mice for pre-clinical research and development will allow an enhancement of diagnostics and drug development, and thereby a more readily available treatment of citizens, reducing unnecessary and expensive medical approaches.
In summary, the EUCOMMTOOLS scientific, societal, and economic impact increased by manifold, tailor-made models for drug screening, validation is made available and to enhance knowledge of gene function and their contributions to health and disease.
List of Websites:
Scientific project coordinator: Prof. Wolfgang Wurst, Helmholtz Zentrum Munich, Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Tel: +49 89 3187 4110
Fax: +49 89 3187 3099
Email: wurst@helmhotz-muenchen.de
Project website: http://www.mousephenotype.org
Part I: EUCOMMTOOLS Executive Summary
The EUCOMMTOOLS (The European Conditional Mouse Mutagenesis TOOLS) Consortium executes conditional gene targeting in murine embryonic stem (ES) cells and establishes Cre-driver transgenic lines for the determination of spatial and temporal gene function.
EUCOMM (FP6) and EUCOMMTOOLS (FP7) are founding members of the International Knockout Mouse Consortium (IKMC, www.knockoutmouse.org) aiming to establish gene mutations for all 20.000 protein coding genes which will build the basis of gene function annotation at scale. The major objective of EUCOMMTOOLS was to complete the conditional gene targeting resource, to establish Cre-driver lines, to further develop conditional tools, and to extend the genetic tool kit for universal use of EUCOMM/EUCOMMTOOLS targeted alleles.
Towards these goals EUCOMMTOOLS has established highly standardized pipelines to reproducibly establish targeting vectors, mutant ES cells and mutant mice under stringent quality control conditions.
In particular EUCOMMTOOLS has established: 1) 2.957 correctly conditionally targeted ES cells, 2) a new conditional targeting strategy for single exon genes, 3) an efficient CRISPR/Cas9 mediated targeting strategy to generate complex conditional alleles, 4) novel universal genetic tools for ectopic gene expression, fluorescence protein tagging, and cell ablation, 5) the evaluation of the risks of site specific mutagenesis approaches, and 6) 223 novel Cre-driver lines and determined partly their Cre-activity.
To date, in a combined effort about 18.500 gene mutations in ES cells have been established. The majority of mutant ES cell lines (about 13.000) have been generated by EUCOMM/EUCOMMTOOLS programs. Based on this resource the International Mouse Phenotyping Consortium (IMPC) has already established approximately 5.000 mutant mice which are currently being phenotyped. The common goal of IKMC and IMPC is to establish an encyclopedia of gene functions serving as reference for the functional annotation of any disease associated allelic variant. The gene targeting vectors and mutant ES cells of EUCOMM/EUCOMMTOOLS are archived by the European Mouse Mutant Cell Repository (EuMMCR, www.eummcr.org) and the mice by EMMA/Infrafrontier (www.infrafrontier.eu). Up to now, more than 1.000 conditional targeting vectors, more than 12.000 mutant ES cells, and more than 1.800 mutant mice have been distributed to academic and commercial users worldwide. Furthermore, more than 1.000 publications acknowledge the use of EUCOMM/EUCOMMTOOLS materials already (source: google scholar).
The success of this ground breaking European programs goes beyond its funding period as EUCOMM/EUCOMMTOOLS have established a self-sustaining repository; the EuMMCR (www.eummcr.org) providing the generated material to academic and commercial researchers also in the future.
Project Context and Objectives:
Part II: EUCOMMTOOLS Context and Objectives
One of the most exciting and valuable scientific challenges is the elucidation of gene function in the context of the entire organism. This elucidation will bring us as close as never before to the understanding of mammalian biology and offer the possibility to fight suffering and disease more efficiently than ever.
Certainly, such an ambitious goal can only be reached in steps. The decoding of the human and mouse genome sequences has been a very important milestone, which resulted in the identification of approximately 20.000 protein coding genes as well as an astonishingly high number of conserved non-coding regions with assumed regulatory functions. The next logical step is to functionally annotate the genome.
This challenge can only be met by comparing gene functions in healthy and hereditarily diseased organisms. Due to embryonic stem (ES) cell technologies, any desired gene mutation can be established in the laboratory mouse, so that all hereditary human diseases can be modeled in this organism. In addition, recent technology developments now allow performing gene mutagenesis in a systematic, high throughput manner to eventually mutate every gene within the mouse genome. This enormous task can only be achieved by an international, coordinated effort, because such a systematic approach will reduce research redundancy, facilitate standardization, and enable the global implementation of technical developments.
In 2007, a worldwide mutagenesis effort began to form which aims at mutating virtually all protein coding mouse genes via gene targeting and gene trapping to speed up functional annotation of the genome. The cornerstones of this International Knockout Mouse Consortium (IKMC) are the European EUCOMM/EUCOMMTOOLS, the U.S. KOMP and the Canadian NorCOMM projects (Collins F.S. et al., 2007a; Collins F.S. et al., 2007b; Skarnes et al., 2011; Bradley et al., 2012; Murray et al., 2012; Donahue et al., 2012, Rosen et al. 2015; www.knockoutmouse.org). The European flagship EUCOMM/EUCOMMTOOLS contributes the largest fraction of mutant mouse genes to the IKMC. Furthermore, EUCOMM/EUCOMMTOOLS was the first project to recognize the tremendous value of conditional mutagenesis (Averx et al., 2004). Based on IKMC, the International Mouse Phenotyping Consortium (IMPC) has been founded to turn the IKMC mutant ES cell resource into mutant mice and determine the mutant phenotype at scale establishing an encyclopedia of gene function. This resource will serve as reference for future disease models.
EUCOMMTOOLS will complete the IKMC resource of mutations for all protein coding genes as far as technically possible. Furthermore, EUCOMMTOOLS will maximize the utility of the conditional IKMC resource by generating up to 250 different, mostly inducible Cre driver mouse lines. In addition, EUCOMMTOOLS will develop novel tools to enhance the versatility of the IKMC resource. EUCOMM’s and EUCOMMTOOLS’ vectors and mutant ES cells are distributed worldwide via the EuMMCR (www.eummcr.org) and the projects’ mice via EMMA (www.emmanet.org). The organizational structure of the EUCOMMTOOLS project is shown in Figure A.
The detailed objectives of the EUCOMMTOOLS program are:
a) to identify 500 genes and their promoters with specific tissue and cell type expression profiles suitable as Cre drivers covering all organs and the major cell types of the body,
b) to establish up to 500 Cre Knockin constructs and recombinant ES cell lines,
c) to establish up to 250 BAC-CreERT2/BAC-Cre driver transgenic mouse lines, and to annotate Cre expression and activity of all 250 Cre driver lines in tissue sections to determine organ and cell type specificity,
d) to provide ‘null mutant first conditionally suitable’ mutant ES cells of approximately 500 miRNA loci. This specific aim was already performed by the Wellcome Trust Sanger Institute, supported by institutional funds. The mutant ES cells have been integrated into the EUCOMMTOOLS resource and are distributed by EuMMCR,
e) to develop gene cassettes for integration into the conditionally mutated EUCOMM alleles such as: cassettes introducing any open reading frame (ORF) of interest, various reporter genes, suicide genes, various recombinases such as Flp, Dre, Cre-ERT2; or siRNAs. These cassettes can easily be integrated into the conditional EUCOMM alleles, either by Recombinase Mediated Cassette Exchange (RMCE) in ES cells, or by TALEN- or CRISPR/Cas-mediated homologous replacement in oocytes,
f) to improve recombinases: to investigate ligand-receptor pairs to improve specificity of recombinases; to develop sequential, conditional switches; to determine position effects of recombination target sites; and to determine recombinase toxicity,
g) to strive towards completion of the IKMC resource by generating up to 3.500 unique, conditionally gene targeted mouse ES cells.
h) to present all EUCOMMTOOLS materials to the public via the EUCOMMTOOLS and IKMC/IMPC web portals,
i) to archive and distribute EUCOMMTOOLS materials via EuMMCR and EMMA,
j) to sustain the EUCOMMTOOLS/EUCOMM and IKMC resource in the long term.
The “Large-scale Integrating Project” EUCOMMTOOLS brings together nine research institutions from five European countries, Australia and USA: the Helmholtz Zentrum München, Munich/Neuherberg, Germany (Prof. Wolfgang Wurst, (EUCOMMTOOLS coordinator), Dr. Joel Schick, Dr. Ralf Kühn (until 06/2014), Dr. Antje Bürger); the Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK (Prof. Allan Bradley, (EUCOMMTOOLS co-coordinator), Dr. William Skarnes, Dr. Vivek Iyer (until 01/2015), Dr. Ramiro Ramirez-Solis); the European Molecular Biology Laboratory (EMBL), Outstation EBI Hinxton, UK (Dr. Paul Flicek, Dr. Gautier Koscielny (until 09/2014), Dr. Terry Meehan); the University of Technology Dresden, Germany (Prof. Francis Stewart, Dr. Konstantinos Anastassiadis); the Consiglio Nazionale delle Ricerche, Monterotondo, Italy (Prof. Glauco Tocchini-Valentini); the Institut Clinique de la Souris, Strasbourg, France (Prof. Yann Hérault, Dr. Marie-Christine Birling); the Universidad de Murcia (Prof. Salvador Martínez); the Medical Research Council (MRC), Mammalian Genetics Unit, Harwell, UK (Prof. Steve Brown), and Human Genetics Unit, Edinburgh, UK (Prof. Richard Baldock), Monash University (EMBL Australia), Melbourne (Prof. Nadia Rosenthal) and the Jackson Laboratory (JAX), Bar Harbor, USA (Dr. Steve Murray). These institutions bring together skills, expertise, resources, and infrastructure to fulfil the ambitious EUCOMMTOOLS goals.
Project Results:
PART III: EUCOMMTOOLS Scientific and Technical Results/Foregrounds
WP1 Gene Selection
WP Leader: N. Rosenthal (MONASH), Partners: A. Bradley (WTSI), Y. Herault (ICS), W. Wurst (HMGU), R. Baldock (MRC/HGU), P.Flicek/G. Koscielny, T. Meehan (EMBL-EBI)
Cre driver selection
WP1.1 has achieved all its tasks. It continued collecting data on publicly available Cre recombinase expressing transgenic mouse lines and presenting them at the CREATE/EUCOMMTOOLS portal (www.creline.org). It also further monitored and incorporated scientists’ suggestions of novel genes for Cre driver production using this portal. For international coordination of Cre driver generation activities, in the last reporting period the Jackson Laboratory was integrated as partner. To avoid duplicated efforts in Cre mouse production, the CREATE website was regularly updated to show the planned and produced resources of the respective Cre driver production projects worldwide including EUCOMMTOOLS. Based on various selection criteria, WP1.1 has identified 732 genes which are suitable for the establishment of specific Cre driver lines. In particular, genes were excluded leading to haploinsufficiency when mutated which is an important aspect when employing knockin strategy (see WP2.1). This list is also presented at the CREATE/EUCOMMTOOLS web portal, indicating also the current Cre driver production status. A regularly updated interface on the CREATE website that displays the produced resources of all the EUCOMMTOOLS Cre lines was created. This is an automatically updated production tracker for each Cre line: As the mice get produced, the production tracker is updated. In addition, a feature was added to the website that provides links from a gene driver to the resulting expression data annotation resource being generated as part of WP6. We have also put in place the necessary informatics infrastructure needed to analyze and display phenotype data from WP7 that will be presented on the IMPC portal: mousephenotype.org.
Gene selection for targeting
The gene list for the EUCOMMTOOLS gene targeting pipeline (WP1.2) contains 3.600 remaining bona fide annotated protein-coding genes. Designs have been generated for all 3.600 genes based on the conventional EUCOMM conditional allele or the artificial intron strategy. Alternative designs have been established for 1.400 genes that failed to produce a conditional targeting vector in the EUCOMM/KOMP-CSD pipelines. Thus, all proper annotated protein-coding genes went through the EUCOMMTOOLS vector production pipeline.
WP2 Vector Construction
WP Leader: W. Skarnes (WTSI), Partners: A. Bradley (WTSI), F. Stewart (TUD)
Generation of conditional KO-first targeting constructs
Since the EUCOMM conditional targeting vectors cannot be used for single exon genes, in WP2.2 a novel strategy was developed by integrating an artificial intron fragment into the conditional gene targeting cassette in the framework of EUCOMMTOOLS. The intron sequence is derived from the Ifitm2 gene, a two exon gene that is highly expressed in all mouse tissues. EST alignments show no evidence for intron retention or cryptic splice sites and the intron is devoid of any conserved elements based on cross-species sequence comparison. A knockout-first allele targeting vector was generated for this gene by KOMP-CSD, and thus we were able to subclone a fragment from the intermediate vector that contains the Zeo-PheS Gateway element imbedded in the intron (see Figure 1). The Ifitm2 gene was cloned by gap repair into a R6K plasmid backbone to generate the template for recombineering (pR6K_Ifitm2i_ZP). To introduce the artificial intron cassette into a BAC, 70-mer oligos are synthesized that contain 20 bp from the 5’ or 3’ end of the intron appended to 50 bp of sequence from either side of the insertion point in the exon of the target gene.
Figure 1. (see attachment)
To validate the artificial intron strategy, we generated targeting constructs for 4 X-linked genes expressed in ES cells (Atrx, Mecp2, Hdac8, and Glp3) by BAC recombineering. The final targeting constructs contain the standard promoterless EUCOMM cassette. Targeted clones were then sub-cloned to ensure they were pure clones and analysed by Western blot for protein levels with commercial antibodies. Only the Atrx gene gave a good signal on a Western, whereas Mecp2 gave a weak signal and no protein was detected with Hdac8 and Glp3 antibodies. The Atrx cells were then transiently transfected with a Flpo expression plasmid to remove the targeting cassette from the artificial intron. As shown in Figure 2, insertion of the artificial intron cassette completely eliminates Atrx protein expression, whereas removal of the targeting element with Flp restores wild-type levels of Atrx protein. These experiments show that the artificial intron strategy is working as desired.
Figure 2. (see attachment)
Corresponding to the new design, quality control (QC) strategies have been developed to validate each step in vector generation. During the whole duration of the project a total of 2.339 targeting vectors have been generated, and recovery attempts were underway for 3.500 projects where a vector is available but electroporation had not been successful enough, i.e. for which less than three ES cell clones are available.
CRISPR/Cas9-assisted targeting
CRISPR/Cas9 sRNA-guided endonucleases may offer an efficient strategy for the recovery of failed conditional targeting projects (Figure 3). Computational analysis has identified suitable CRISPR sites at the 3’ loxP site of the targeting vector for 90% of the failed projects.
Figure 3. (see attachment)
Over the past year, we generated gRNA plasmids for 88 failed genes and tested the efficiency of Cas9-assisted targeting as a recovery strategy. We reasoned that many of the failed projects may be due to difficulties with LR-PCR genotyping across the long homology arms, rather than a failure to target the desired locus. Therefore, we have also developed a process to shorten the vector arms based on linear:linear ET cloning recently described by Francis Stewart (WP3) and colleagues (Fu J et al., Nature Biotechnology 2012).
Vector Shortening Process to Facilitate Cas9 Assisted Targeting Route
Conventional conditional vector targeting failures in the main EUCOMMTOOLS pipeline can be due to a combination of two problems, low targeting frequency and difficulties in LR PCR genotyping. While Cas9-assisted targeting can address the former issue, it does not help resolve the latter. As Cas9-assisted targeting requires much shorter targeting arms (1kb or less) than the conventional route, it would also be optimal to shorten the 5kb targeting arms found in EUCOMMTOOLS vectors to 1kb or less to facilitate genotyping and improve its success rate. To that end, we have developed a high throughput process for shortening the arms of EUCOMMTOOLS conditional gene targeting vectors in a 96 well format (Figure 4). The process is based on linear-linear recombineering using the recET recombineering system (Fu et al., Nature Biotechnology 2012).
Figure 4. (see attachment)
We have this process operating in a 96 well format and, to date we have generated 242 shortened vectors. These vectors, along with the corresponding gRNA plasmids, have been transferred to WP4.2 for ES cell production.
In summary, from 3.750 gene designs we have successfully constructed 2.339 targeting vectors including 330 vectors for CRISPR/Cas mediated targeting. In addition 1.679 vectors were recovered from failed project. Thus in total we have provided 4.018 vectors for electroporation in ES cells.
Cre-BAC knock-in vectors for recombination-mediated cassette exchange at a pre-defined locus
The goal of WP2.1 was to construct the vectors for 500 BAC-CreERT2/BAC-Cre transgenic ES cell lines. The project’s primary approach towards the objective of this work package was to use recombinase mediated cassette exchange (RMCE) as a means to integrate single-copy BAC-Cre recombinase transgenes at pre-determined loci (ROSA26 and HPRT) in ES cells. It was previously reported that for the site specific integration of BAC transgenes the efficiencies of transgenesis were too low to be used for pipeline production. During pilot studies the HPRT landing pad integration failed to yield any BAC integrants and the ROSA26 landing pad integration produced very low rates of integrated BACs.
The low efficiencies of site specific BAC transgenesis in the C57BL/6N ES cells that we have experienced led to the suggestion that an alternative approach might be used. In this alternative approach the BAC would be modified by insertion of piggyBac transposon inverted repeats within the BAC vector backbone in close proximity to the genomic DNA cloning sites. It has been demonstrated that by pronuclear injection of a mixture of piggyBac transposase mRNA and the modified BAC a high level of single copy, randomly integrated BAC transgenes ca be achieved in single cell embryos, which can then develop to make transgenic mice. This technology allows the direct production of transgenic offspring, circumventing ES cell and chimera generation.
The gene specific modifications of the BAC inserting the EGFP-CreERT2 are performed exactly as for the previous (site specific integration) pipeline. Following the gene-specific modification the production pipelines diverge as there are two further recombineering steps that insert piggyBac 5’ and then 3’ inverted repeats at either ends of the BAC vector backbone.
The BAC constructs have been sent to mouse production centres, to generate transgenic offsprings. After generating 15 transgenic lines it turned out that the vector was not stable as shown by restriction enzyme mapping and finally by DNA sequencing. Therefore, this strategy was abandoned.
As an alternative strategy for the BAC-CreERT2 approach, in parallel we have developed a working pipeline for the construction of Cre recombinase knockin targeting vectors involving the re-cycling of intermediate vectors made for IKMC projects (EUCOMM, EUCOMMTOOLS, KOMP) exploiting their modular Gateway capacity. No new recombineering based vector construction is involved, just a single high efficiency vector assembly step. The major components, a gene specific intermediate vector and a reading frame specific nlsGFP/CreERT2 knockin cassette are shown below (Figure 5):
Figure 5. (see attachment)
Intermediate vectors are re-arrayed onto 96 well plates, and their identity is confirmed by DNA sequencing with a >95% success rate. The resulting validated intermediate vectors are then put through a three way Gateway reaction (intermediate vector, Cre cassette and DTA containing plasmid backbone), and multiple colonies are analysed by DNA sequencing. The integrity of the resulting Cre knockin final vectors is confirmed by a combination of physical (gel of linearised plasmid DNA) and DNA sequencing (gene identity, reading frame of CreERT2 cassette). The efficiency of this process was 100% with 457 final vectors assembled to date. Vectors that failed targeting with the PGK-puromycin version were reassembled with the Beta-actin neo cassette (which targets at a higher success rate) to rescue the projects. Successful clones were re-arrayed onto 96 well plates for electroporation into JM8 ES cells.
Furthermore, within this workpackage, the EUCOMMTOOLS consortium has commenced a pilot experiment Figure 6) to test the possibility of expedited engineering of Cre knockin mice directly in mouse zygotes exploiting the CRISPR/Cas9 system. This would create an alternative and speedy route to Cre knockin mice for the Cre knockin mouse generation project.
Figure 6. (see attachment)
Vector shortening for CRISPR-assisted targeting in mouse zygotes
In cases where CreERT2 knockin vectors have not yielded efficient targeting in ES cells even after repeated electroporations there is a possibility of rescuing the project by the Cas9-assisted targeting route in mouse zygotes. However, to optimize the probability of success of this strategy the following modifications of the targeting vectors must be performed:
1) the vector must not contain gRNA recognition sites so it should not be cleaved by Cas9 nuclease
2) shortening the targeting arms from 5kb to 1kb should improve genotyping
3) CreERT2 cassettes no longer requires a mammalian drug selection marker
Employing the linear-linear ET recombination vector shortening process described in the WP2.2 section, the intermediate vectors for 48 EUCOMMTOOLS Cre genes which have definitively failed targeting are being converted to short arm vectors suitable for Cas9-mediated targeting in mouse zygotes (Figure 7).
Figure 7. (see attachment)
The intermediate vectors that enter the process have been previously sequence validated and only require re-arraying to commence the vector shortening process. Pairs of U6 gRNA plasmids with minimal off target signatures to sequences in the deleted genomic region are also produced. A version of the standard EGFP_CreERT2 cassette in which the mammalian selection marker has been deleted by exposure to Dre recombinase in Dre expressing E. coli is used for the Gateway vector assembly reaction.
CRISPR/Cas9-Assisted Targeting directly in mouse zygotes for making Cre Knock-in mice
In collaboration with Steve Murray’s group at the Jackson laboratory, we were building 48 short arm targeting vectors tailored for use in mouse zygotes using a Gibson assembly strategy. This route can be used to construct alternate alleles such as 5’ ATG knock-ins where the endogenous start codon (ATG) is replaced with a Kozak ATG version of the same EGFP_T2A_CreERT2 cassette used in the EUCOMMTOOLS high throughput Cre knockin pipeline. There is no need for a selection marker cassette for vectors destined for direct injection into the mouse zygote. The Gibson assembly vector assembly strategy shown in Figure 8 combines:
1) PCR amplification of 1 kb targeting arms from C57BL/6 genomic DNA
2) a custom R1R2 Gateway Cassette with Zeocin resistance
3) an Amp resistant plasmid backbone
Figure 8. (see attachment)
Thus, this technology paves the way for next generation Cre-driver line production.
In summary, we have generated 55 BAC- CreERT2 vectors, 447 CreERT2 Knock-in vectors and 12 short arm CreERT2 Knock-in vectors for CRISPR/Cas9 mediated targeting in zygotes. In total, we have produced 514 CreERT2 targeting vectors.
WP 3: Research, Technology and Reagent Development
WP Leader: F. Stewart (TUD), Partners: A. Bradley (WTSI), R. Kühn (HMGU), W. Skarnes (WTSI), W. Wurst (HMGU)
Establishment and validation of an IKMC toolkit
The focus of WP3 was the development of new technologies to optimize the utility of the conditional EUCOMM/TOOLS and IKMC resources. In particular, universal cassettes (for reporters such as YFP and LacZ and recombinases such as EGFP-CreERT2) have been produced (WP3.1).
Construction of a universal cassette for retargeting/RMCE.
We have developed a series of dual-purpose universal cassettes for the introduction of fluorescent proteins and a variety of other reporters/recombinases into EUCOMM(TOOLS)/KOMP genes by facilitated gene targeting or by recombination-mediated cassette exchange (RMCE). Each of these constructs is flanked with attL1/L2 sites such that they can be assembled into targeting vectors in an in vitro Gateway reaction with Gateway-adapted intermediate vectors from the EUCOMM(TOOLS)/KOMP resource (see Figure 9).
Figure 9. (see attachment)
The universal cassettes contain site-specific recombination sites (5’ Frt and 3’ loxP) for dual recombinase dRMCE (Osterwalder M., et al. (2010), Nat Methods 7(11):893-5) in targeted ES cell clones. Expression of the reporter is dependent on splicing and the production of a fusion protein. Therefore, we engineered these cassettes in each of the three reading frames as well as a Kozak-ATG version for expression from non-coding exons. For optimal expression of the reporter, we included a novel 2A-like sequence (LF2A) which our experimental evidence (presented in last year’s report) suggests can be used efficiently in fusions to both secreted/transmembrane and non-secreted proteins.
Construction of a universal targeting cassette.
The activity of the lacZ gene is compromised in fusions to genes that encode N-terminal signal sequences (Skarnes WC, et al. Proc Natl Acad Sci USA. 1995 Jul 3;92(14):6592-6). For the EUCOMM project, we used a promoter-targeting cassette that contains an internal ribosome entry site (IRES) to initiate translation of the lacZ reporter. Expression of lacZ is frame independent and should work irrespective of protein class. However, efficient use of the IRES sequence is known to be influenced by local sequence content and thus may not be reliable in all genes. To explore the L2A sequence further, we constructed promoter-driven targeting cassettes that contain this sequence fused to the 5’ end of lacZ (see Figure 10) and carried out gene targeting experiments for several non-secreted and secreted loci. In these experiments, we included promoter-driven targeting cassettes that contain the IRES sequence for comparison. Based on the intensity of Xgal staining in cells, the LF2A cassette showed equal or higher levels of staining for both secreted and non-secreted targeted loci compared to the IRES cassette. Now reading frame specific LF2A targeting cassettes are routinely used in the construction of most EUCOMMTOOLS conditional targeting vectors
Figure 10. (see attachment)
Construction of Cre cassettes.
At the EUCOMMTOOLS meeting in Alicante in April 2011, it was agreed to pursue Cre knock-ins into a selected set of target genes and to compare this method to RMCE of Cre-BACs into Hprt and Rosa26. Subsequently it was decided to develop a full scale production pipeline of targeted Cre knockins described in WP 2.1. To this end, and with input from the EUCOMMTOOLS Scientific Advisory Board, we developed dual eGFP reporter/inducible Cre recombinase (CreERT2) cassettes that are used for knock-in strategies, primarily re-targeting of Gateway assembled pre-existing intermediate vectors that also can be used for dRMCE. Co-expression of eGFP provides a cellular marker for CreERT2 expression, facilitating the characterisation of Cre protein expression by direct fluorescence or by immunohistochemistry with anti GFP antibodies. The vectors now used are shown in Figure 11. Initially a PGK puromycin selection cassette version was developed for RMCE (standard conditional IKMC cells are neoR). However, these vectors delivered less efficient targeting compared to standard EUCOMMTOOLS conditional alleles, so an alternate Beta-actin-neo version was built and is now used in the Cre Knock-in targeting pipeline.
Figure 11. (see attachment)
The functionality of tamoxifen inducible Cre activity and fluorescent protein expression was tested in an ES cell line containing a floxed lacZ reporter in a ubiquitously expressed gene (Arid1a).
The first crosses of Cre knockin mice for the secreted Gpx3 indicate robust, tamoxifen inducible, activity of Cre on a floxed lacZ reporter in the ROSA 26 locus in vivo, demonstrating the full functionality of the vector in vivo.
A vector for the knock-in of EGFP_2A_CreERT2 into the 3’ UTR of endogenous genes (IRES/nlsEGFP/CreERT2) has also been developed. This vector should avoid potential haploinsufficiency associated with knock-in alleles. De novo recombineering/Gateway Vector assembly for a set of 24 genes resulted in 19 final IRES_nlsEGFP_CreERT2 3’ knockin vectors which have been targeted in ES cells. As this route involves new recombineering of targeting vectors, it does not represent a solution for accelerating the production of EUCOMMTOOLS Cre knockin mice, but the alleles are of interest as a comparison to other Cre knockin strategies and for cases of potential haploinsufficiency.
Other EUCOMMTOOLS cassettes.
EUCOMMTOOLS cassettes have been classified into cassettes which can be used with existing off-the-shelf IKMC intermediate vectors, 3’ IRES containing vectors for insertion after the termination codon of the endogenous protein and 5’ KATG (Kozak-ATG) cassettes used to replace the initiating methionine; the latter two approaches require custom recombineering approaches utilizing EUCOMMTOOLS vector components. Modular cloning approaches allow re-cycling of validated vector components into these 5’ and 3’ cassettes. The list includes cassettes for a variety of fluorescent proteins (presently being incorporated into the modular vector system are far red, orange and blue fluorescent proteins), chromosome engineering that allow fluent replacement of rearranged sequences, multimodal reporters employing 2A sequences (fluorescent, luminescent, MRI), and neutral surface reporters for isolating cell populations (developed in collaboration with Timm Schroeder, HMGU Munich), and a 3’ protein tagging system.
Table 1. (see attachment)
One of the cassettes we have developed is a tri-modal reporter which combines fluorescence, luminescence and PET imaging (HSVtk) reporters as shown in Figure 12. It will be employed to track transplanted mouse and human stem cells in animal models of human diseases. All three reporter activities have been validated in mouse ES cells with CAGGS PiggyBac integrations of the triple reporter in collaboration with Sanjay Sinha’s group at the Laboratory of Regenerative Medicine, Cambridge.
Figure 12. (see attachment)
Several of the EUCOMMTOOLS cassettes have also been validated by gene targeting human ES cells (H9 and H1) in collaboration with Prof. Roger Pedersen’s laboratory in Cambridge and thus are also of value in engineering human as well as mouse stem cells.
We have also developed a new cassette which has been employed in the conditional disruption of long non-coding RNAs (lncRNAs) in collaboration with Prof Adrian Bird’s laboratory in Edinburgh. This cassette contains elements to truncate both spliced and unspliced lncRNA transcripts using Rabbit beta-globin exon/intron and repeated SV40 polyA sites with site specific recombination sites to enable a conditional strategy which involves either deletion of the promoter or truncation of the transcript. It has now been employed in constructing CKO vectors for more than 50 lncRNA targets. The disruption cassette’s structure is shown below Figure 13):
Figure 13. (see attachment)
The development of neutral ligands for mammalian SSR-LBD applications
To improve the function of the CreERT2 estrogen-ligand binding domain, a mutant library of estrogen binding domains (Cre-EBD) has been created. Its screening protocol has been optimized (WP3.2). We decided, however to prioritize WP3.3 to enhance Cre-driver production first.
Cre-EcBD (ecdysone BD) fusion proteins were built and functionally evaluated. Previously the Stewart lab evaluated the ecdysone BD in similar experiments to conclude that the EcBD did not convey repression onto Cre recombinase in the absence of ligand (unpublished results). Further experiments, performed in ES cells with a positive control conclusively reinforce the previous conclusion – the EcBD does not inhibit fusion proteins in cis. Hence we discontinued EUCOMMTOOLS experiments with the EcBD. The development of a new recombineering method for codon-directed, site-specific mutagenesis has been successful. Using this method, a very high quality site directed mutant library of the estrogen binding domain (EBD), with codon-based mutations focused on 36 amino acids around the ligand binding pocket, has been made. Furthermore, the yeast shuttle vector for expression of the mutant EBD library has been finalised. A pilot experiment with a FLP-wtEBD showed satisfactory properties of recombinase repression before, and recombinase activity upon, ligand induction. After removal of a restriction site, the final yeast shuttle vector for the molecular evolution experiments has been completed. Pilot experiments in yeast with FLP-wtEBD encountered problems with low transformation efficiency. This bottleneck has now been solved and high transformation efficiency has been obtained to introduce more than 10.000 mutant EBD clones into yeast for selection. We have established mutant Flp-EBD yeast library for selection of improved EBD ligand binding. However, it turned out that the vector containing strain exhibit basal Flp activity without ligand binding. Thus, the current yeast strains are not suitable to screen for improved flp activity upon ligand binding due to the basal flp activity. Also Cre shows basal activity in yeast as well and therefore Cre-EBD is also not suitable to improve EBD in this yeast strain. Therefore, we are testing different strains and conditions to overcome this problem beyond funding of this project.
The development of type 3 conditional switches, evaluation of limits of conditional mutagenesis and nuclease technologies
In WP3.3 type 2 switches using Flp and Dre have been successfully implemented in vitro and in vivo. To evaluate potential problems and limitations of conditional mutagenesis, a series of various recombinase expressing vectors and reporter cassettes have been generated, to determine site specific recombinase (SSR) activity as well as toxicity in different species (WP3.4). We could show that SSR toxicity is dependent on SSR activity independent of the location of cryptic sites across the genome. It turned out that there is a hierarchy of SSR toxicity Cre>Dre>Flpo (WP3.4). In the framework of WP3.5 innovative, sequence-specific TALENs and CRISPR guide RNAs against loxP and Frt sites have been constructed. TALENs are fusion proteins of a customized DNA binding domain made of TAL repeats and the FokI nuclease domain whereas CRISPR represents sequence specific guide RNA to which Cas9 nuclease is binding in both cases leading to sequence-specific DNA binding and to site specific double strand breaks within the binding domain. The TALEN/gRNAs has proven highly active in vitro. In addition, an improved standard protocol for the generation of mutants by zygote microinjections of TALEN and CRISPR/Cas9 has been developed and published (Wefers et al. 2013, Schick et al., in revision). Funding for this WP expired after 3 years. Overall the objectives have been achieved, albeit with some adaptations from the original plan by integrating the CRISPR/Cas technology which was not available at the time of application. In addition, this WP includes two lines of exploration that are appropriate to the general goals of technological development for EUCOMMTOOLS but were not defined in the original proposal. These two additional avenues are:
(a) the use of transposition to achieve BAC transgenesis using both PiggyBac and Sleeping Beauty transposons (Bradley and Stewart labs);
(b) the use of designer nucleases to promote targeted mutagenesis and homologous recombination (Skarnes, Kühn/Wurst and Stewart labs).
These extra objectives are based on recent developments in the technical field and were incorporated because they are highly relevant to WP3 goals (Wefers et al. 2013, Schick et al., in revision). Part of this WP will continue after funding.
In summary, WP3 has achieved its goals taking into consideration adaptions due to technical obstacles or novel developments which we integrated smoothly into our research program.
WP4 Embryonic Stem Cell Production
WP Leader: W. Wurst (HMGU), Partners: A. Bradley (WTSI), B. Skarnes (WTSI)
Generation of BAC-CreERT2-T2A-eGFP/Cre-T2A-eGFP transgenic ES cell clones
It turned out that the Cre-ERT2 knock-in strategy taking advantage of EUCOMM intermediate vector resource was the most efficient and stable strategy to establish Cre-drivers at scale. We have established 493 Cre targeted ES cells representing 378 unique genes using this approach. The original proposed BAC-Cre RMCE (recombination mediated cassette exchange) strategy in Hprt or Rosa locus did not result in stable, germline competent ES cell clones by testing 55 different vectors. Therefore, we entirely focused on Cre-knock-in strategy. Based on an efficiency of 60% germline transmission, the injection of 2 clones for each of the 378 targeted genes allowed us to meet our goal of producing 223 strains of Cre knock-in mice (Figure 14). In addition, about 20 vectors are still in production using CRISPR/Cas mediated knock-in in zygotes and 16 in germline testing.
Figure 14. (see attachment)
Thus, we have established sufficient CreERT2 expressing targeted ES cell lines to generate 250 CreERT2 transgenic driver lines.
Production and QC of 3.500 conditional knock-out ES cell lines
In total, in the course of EUCOMMTOOLS 4.501 ES cell electroporations were performed comprising 3.618 genes. All proper annotated genes were processed. We have correctly targeted 2.565 protein coding genes and in addition 392 miRNA genes. In total, 2.957 genes were targeted. Some of the remaining failed genes are not amenable to current mutagenesis technologies. Therefore, we have begun to employ CRISPR assisted mutagenesis using conditional EUCOMMTOOLS targeting vectors. Testing 254 conditional targeting vectors we achieved correct targeting of 146 (58% success rate) genes that previously did not give targeted products. At this point, it can already be concluded that not all genes will be targetable using current conditional strategy and that simple CRISPR/Cas mediated deletion alleles need to be generated for the remaining genes. However, this would also require development and validation of a separate production pipeline.
In summary, we have generated additional 2.957 target genes and have adapted CRIPSR/Cas technology to generate conditional targeted genes by re-using EUCOMMTOOLS targeting vectors.
WP5 Generation of Transgenic Mouse Lines
WP Leader: R. Ramirez-Solis (WTSI). Partner: Y. Herault (ICS), G. Tocchini-Valentini (CNR), N. Rosenthal (Monash/Jax), Steve Murray (Jax), and W. Wurst (HMGU)
Generation of Cre-driver knock-in transgenic mouse lines
The WP5.1 Cre mouse production centers have generated 223 (reflecting 221 genes, 36 lines are still in the pipeline) Cre driver mouse lines by injecting 318 unique Cre-driver ES cells. These lines are crossed to reporter lines (lacZ, tdTomato) to monitor Cre activity in vivo. Especially the last year has seen enormous progress in the production of new Cre-driver knock-in transgenic lines from the all the EUCOMMTOOLS mouse production centres. At the last count just prior to writing this report, 223 transgenic mouse strains have been produced for 221 unique Cre-driver loci. This represents about 88% of our stated goal. A further 16 strains are in the breeding process to achieve germline transmission. The partners ICS, WTSI and CNR have delivered their original requirements (ICS 49/50. WTSI 107/100, and CNR53/50). The two new partners (Monash and Jax) have integrated successfully into the workflow and are raising the total number of lines produced already. These partners currently inject 20 vectors directly into zygotes taking advantage of CRISPR/Cas9 mediated knock-in strategy (which are not included into table 2. The table illustrates the detail of the total number of injections, new strains (# Genes GLT) generated, and strains still in process at each of the participating centers (Table 2).
Table 2. (see attachment)
Table 3. (see attachment)
The next step in the process was to breed the Cre-driver knock-in lines to Cre-reporter strains. When the project started, the chosen reporter line was ROSA26-loxP-STOP-loxP-lacZ. During the development of the project based on SAB suggestion, a decision was made to switch the reporter strain to a tomato fluorescent reporter, namely B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze which provides better cellular resolution. The final proportion of strains on the different reporters is illustrated in the table 3 below. At last count, overall, 58% of the strains were crossed to the lacZ reporter and 42% to the fluorescent tomato reporter.
In addition, SOPs for tamoxifen treatment, perfusion and dissection have been established for CreERT2 transgenic lines together with WP6. Double heterozygote animals (het for the Cre-driver, and het for the reporter locus) were dosed with tamoxifen as follows:
• Adults
• P56 (+/- 3 days) mice were IP injected, once a day for 5 consecutive days and perfused 8 -12 days after the first injection. Each mouse received 100µl (10mg/ml) of tamoxifen solution per day.
• Pups
» P6 (+/- 1 day) mice are dosed with 1 sub-cutaneous injection of tamoxifen solution and perfused at P14 (+/- 1 day). Pups receive 20µl of tamoxifen solution per gram of body weight.
Originally, for each strain 2M and 2F were injected with tamoxifen, including controls. This arrangement was revised at the May 2014 in Rome EUCOMMTOOLS meeting. These are the revised number of samples for each treatment per strain:
» No control pups shipped
» 1 Adult p56 (male) + back up (preferably male, since no gender effects on expression had been observed)
» 2 pups P14 (male + female) + back up (either sex).
Since many mutant mouse strains were made with the drug-resistance cassette still present at the locus in order to avoid the negative effect of the Dre-treatment on the ESC germline capability, four strains were generated with and without Dre-excision of the selectable cassette in comparison. This allowed the evaluation of the effect of the marker on the specificity of expression of the Cre-driver. The Cre-driver loci for the strains Cpne6, Gpx3, Itih1, and Prdm16 loci were chosen. Mutants for both (excised and non-excised) were bred to reporter strains, and double heterozygotes animals were treated as usual and shipped to WP6 for annotation. No differences in Cre-activity have been observed.
Quality control of conditional mutant ES cells
WP5.2 served as quality control of produced ES cells in respect to germline transmission efficiency. In contrast to the originally proposed 50 transgenic mouse lines, more than 5.000 mutant mice have been established using EUCOMM/EUCOMMTOOLS ES cells generated by WP4.2 as well as by EUCOMM/KOMP. The mouse production was mostly performed by IMPC members. Thus, quality control of the ES cell resource has been provided extensively. All transgenic mice established in the framework of EUCOMM/EUCOMMTOOLS are archived for distribution by EMMA/Infrafrontier (www.infrafrontier.eu). Some of the Cre-driver lines will be in addition available via the Jackson Laboratory (www.jax.org).
In summary, the establishment of Cre-driver lines and conditional target mouse strains has been achieved.
WP6 Cre Expression Annotation
WP leader: S. Martinez (UM)
WP6 has developed a reference atlas of anatomical structures at P7, P14 and P56 mouse to annotate Cre expression at the cellular level. An ontogenic annotation trees have been developed for standardized nomenclature. We have established adequate and reproducible conditions to get perfect high quality sections for histological analysis. The whole bodies of P14 mice have been processed in sagittal and transversal sections. Twenty sagittal sections have been processed in different series to detect lacZ or tdTomato expression and GFP by immunostaining in combination with specific cell markers. All the body organs are represented in sagittal sections, which gave us an overview of the general expression pattern. These expression patterns have been compared with publicly available expression data bases (Eurexpress, Genepaint, Allen brain project).We have generated an expression maps in relation to the reference atlas. According to the suggestions of the SAB WP6 focused on the analysis of 1 male, 1 female at P14 and 1 male at P56 offsprings to enhance annotation efficiency. At present, tissues of 193 Cre-driver stains have been obtained. Among them, 74 Cre-driver lines expression annotations have been achieved: Tns1, Krt17, Acan, Mb, Tshb, Prdm16, Cpne, Ucp1, Gpx3, Heatr8, Tle6, Trpv1, Smyd3, Hoxa2, Tac2, Pou2f1, Rbpjl, Spp2, ROSAL2/WT, Ptprcap, Cd3e, Atp6v1g2, Ccl25, Hes5, Myo1a, Insl3,Csk/Csktm1, Dmbx1, Mia, Spink8, Entp5, Acsm4, Dbx1, Cdx2, Gnai2, Pkd2l1, Sfrp1, Cnp, P2rx7, Pdgfrb, Gast, Cd63, Cpne6, Nefh, Amhr2, Rbfox3, Wnt9a, Barhl1, Zfp819, cldn7, Sgca, Pbx2, Zbtb32, A830010M20F, Lztfl1, Ttl25, Sds, Aqp3, Eomes, Spic, Zfp629, Slc26a5, Fga, Tbx2, Tff1, Cmtm5, Ttr, Myh4, aldh3a2, Igj, Slc26a4, Mog, Cdh2, and Sost.
A total number of 85.577 annotations at the body and brain have been performed resulting in expression annotation to 15.082 cell types.
All brain and body annotations together with selected images are published on our online platform at www.imib.es/AnotadorWeb. The following images show representative examples of Cre-driver (Tomato) lines annotated from body (Figure 15) and brain sections (Figures 16, 17) representing the expression of the endogenous genes to almost 100%.
Figure 15. (see attachment)
Figure 16. (A, B) (see attachment)
Moreover, the analysis of the expression of the new reporter line based on a fluorescent protein reporter (Tomato) clearly shows an advantage for the annotation of specific type of cells in the brain (Figure 17).
Figure 17. (see attachment)
Taken together, 74 lines have been annotated. Thus, we are behind the original plan date because of the delay in CreERT2 ES cell provision based on changing strategies from BAC- CreERT2 to Knock-in CreERT2 lines
WP7 Phenotyping of Cre Transgenic Mice
WP Leader: S. Brown (MRC)
In WP7 BAC-CreERT2 transgenic lines were planned to be phenotyped to determine potential by-stander effects of additional genes located on the BAC. Since we changed strategy towards knock-in into the endogenous locus and pre-selected promoters/genes showing no haploinsufficiency when mutated we did not expect phenotypic alterations in these transgenic mice. Nevertheless, eight Cre-ERT2 transgenic knock-in lines have been selected and six lines were imported by partner 8 and have been assessed for phenotypic consequences of Cre expression and of potential haploinsufficiencies. To date, phenotyping of lines Gpx3, Ucp, Mb and Tshb have been completed. As expected, due to the change of strategy no phenotypic alterations were found after intensive primary screening in the Harwell mouse clinic (see also IMPC phenotyping pipeline) compared to wildtype littermates. This work is performed exclusively by institutional funds of MRC Harwell.
Taken together, phenotyping of Cre-driver lines did not result in phenotypic alterations.
WP8 Bioinformatics
WP Leader: B. Skarnes (WTSI), Partners: S. Martinez (UMH), W. Wurst (HMGU)
WP8 (Bioinformatics) was supporting the design, tracking and reporting of eGFP-CreERT2 vectors, of the artificial intron containing conditional targeting vectors of EUCOMMTOOLS, of CRISPR/Cas mediated targeting as well as data tracking within the consortium. In addition, it is integrating data and maintaining the EUCOMMTOOLS/IKMC/IMPC web portal. The production of Cre knockin ES cells based on EUCOMM intermediate vectors has been tracked in our main production LIMS and the production of Cre knockins in mice is tracked in iMITS. All of the information on Cre knockin ES cells as well as mouse production have been uploaded into iMITS. iMITS has been migrated to the EBI, Hinxton where the data is archived and displayed on the IMPC website (www.mousephenotype.org). The informatics activities of the IKMC - which includes EUCOMMTOOLS - have now been transferred to the IMPC for long term sustainability reasons. The project website can always be accessed via http://www.eucommtools.org which continues to direct the user to the EUCOMMTOOLS project website, now embedded into IMPC website. WP8 has achieved its goals by supplying all actual bioinformatics support needed by the production work packages.
WP9 International Coordination and Databases
WP Leader: N. Rosenthal (MONASH), Partners: A. Bradley (WTSI), W. Wurst (HMGU)
The goals of the EUCOMMTOOLS International Coordination Committee (ICC) were to promote international collaboration and the adoption of common standards and committed to the project. We included stakeholders from around the world, coordinated activities between the existing EU coordination action CREATE (dev.creline.org) and the major non-European Institutions building similar resources, including NIH (US), NINDS (US), Scripps Institute (US), JAX/IMSR (US), Allen Institute (US), RIKEN (Japan), MSHRI (Canada) and APN (Australia). The aim of WP9 was to align activities between centres, to assimilate their recommendations and reports, and to continue developing a technology roadmap for mouse conditional mutagenesis and generation of human disease models.
Members of the ICC (and corresponding Cre driver related initiatives) have been:
• Nadia Rosenthal (Monash University, Australia, CREATE consortium coordinator)
• Colin Fletcher (NIHGRI, USA) (KOMP, Cre Driver Network)
• Janan Eppig, Steve Murray (The Jackson Laboratory, USA)
• Colin McKerlie, Lauryl Nutter, Marina Gertsenstein (TCP, Canada) (NorCOMM2ls)
• Elizabeth Simpson (UBC, Canada) (CanEuCre)
• Geoff Hicks (U Manitoba, Canada) (NorCOMM, NorCOMM2ls)
• Andras Nagy (MSHRI, Canada) (NorCOMM2ls, Cre-X-Mice)
• Hongkui Zeng (AIBS, USA) (AIBS Transgenic Programme)
• Kent Lloyd (UC Davis, USA) (KOMP, MMRRC)
• Michael Dobbie (Australian Phenomics Facility - APF, Australia)
• Nat Heintz, Laura Kus (Rockefeller U, USA) (GENSAT)
• Yuichi Obata, Atsushi Yoshiki ((RIKEN-BRC, Japan) (RBRC)
WP9 partners organized meetings in the framework of EUCOMMTOOLS, IKMC and IMPC meetings (July 5-7, 2011: EUCOMMTOOLS Progress Meeting (Rome, Italy); December 2-4, 2013: INFRAFRONTIER-IMPC-IKMC Meeting and EUCOMMTOOLS workshop (Rome, Italy); October 26-30, 2014: MGI 25th Anniversary Celebration at The Jackson Laboratory (Bar Harbor, USA)); as well as ICC conference calls agreeing on common nomenclature and SOPs for generating and annotating Cre drivers, implement web services in order to link the respective databases, disseminate conclusions and SOPs, develop plans for long term sustainability and distribution of the resource as well as emerging technologies for Cre driver generation relating to gene editing.
WP 10 Distribution and Archiving
WP Leader: A. Bürger (HMGU)
The WP10 objective is to import, expand, quality control, archive and distribute all EUCOMMTOOLS vectors and mutant ES cells via the EuMMCR (European Mutant Mouse Cell Repository). EuMMCR was founded as the EUCOMM Material Distribution Unit, which archives, distributes, and quality controls EUCOMM and EUCOMMTOOLS materials, i.e. conditional gene trap and conditional gene targeting vectors as well as EUCOMM gene targeted and gene trapped ES cells. Distribution is performed internally (to EUCOMM and EUCOMMTOOLS mouse production centers) and externally, to the scientific community worldwide. EuMMCR has established its own website (www.eummcr.org) which informs the user about the available resources and the ordering procedures and ensures a better and easier access to and visibility of the projects’ resources.
ES cell distribution: Upon orders from interested scientists worldwide, the EuMMCR lab is expanding requested ES cell clones and refreezing those clones in several aliquots, which allows EuMMCR shipping aliquots to the requesting scientists and maintaining the resource. Furthermore, the EuMMCR lab is performing quality controls on the ES cell samples like e.g. mycoplasma test, verification of the identity of the mutated allele (PCR), development of a genotyping protocol (PCR), verification of the correct allelic integration by long range PCR (LRPCR) over the 5’ and 3’ homology arms of the targeting vector, and counting the copy number of neo and lacZ integration (by qPCR) to exclude double or multiple integrations of the targeting cassette.
Import of EUCOMMTOOLS resources: More than 16.500 conditional EUCOMM/EUCOMMTOOLS vectors were imported from partner 2. The vectors have been delivered in 96well and 384well plates as glycerol stocks. Upon arrival the vector plates were immediately stored at -80°C. The EUCOMM and EUCOMMTOOLS ES cell clone collections in the EuMMCR storage facility is currently composed of approximately in total 2.3 Mio samples.
EuMMCR production and distribution numbers: EuMMCR production started in 2008. The production and distribution numbers from 2008 until September 2015 (expected numbers until end of 2015 are shown in brackets) are shown in Figure 18. EuMMCR has distributed a total of 11.903 (12.098) ES cell clones to the international scientific community for transgenic mouse production. Of these, 9.441 (9.636) ES cell clones were delivered since the beginning of EUCOMMTOOLS. The differences in distribution to production numbers are due to quality control failures, which are not sent out to the end user.
Vector distribution started in 2009 and has now reached a total of 926 (959) vectors as shown in Figure 19. Overall the number of requests (not shown) and distributions show that the EUCOMM and EUCOMMTOOLS resources are well received in the scientific community worldwide.
Figure 18. (see attachment)
Figure 19. (see attachment)
mirKO resource: The Sanger Institute’s mirKO resource is a collection of targeting vectors and heterozygous targeted miRNA genes in JM8.A3 ES cells using a gene deletion strategy. In total, 212 targeted alleles (clusters and singleton knockouts) corresponding to 253 different miRNA genes have been sent to EuMMCR for archiving and stored in liquid nitrogen storage and are ready for distribution upon requests. Each microRNA gene is represented in the archive with up to 6 individual ES cell clones. The resource was published (Prosser HM et al. Nat Biotechnol. 2011; 29(9):840-5.).
Cre-ERT2 Knockin ES-Cell resource: A large collection of 375 recombinant ES cells have been established and archived. The EuMMCR imported this very important resource from EUCOMMTOOLS partner 2 (WP 4.1) into our archive, as well as the corresponding data. Thus, Cre-ERT2 vector and cell resource is also available to the scientific community. Up to now, we imported more than 28.000 samples from these Cre-Driver lines.
Strategic partnership with genOway: EuMMCR recently entered a strategic partnership with genOway. This allows us to distribute all products available at EuMMCR to “for-profit” organizations. Industry scientists benefit from stringent genOway quality controls and the unmatched reliability of its platform in the production of the EUCOMM and EUCOMMTOOLS conditional KO models for commercial partners. For more information please refer to http://www.genoway.com/products-services/eucomm/eucomm-conditional-knockouts.htm
Ordering process: The ordering process has been re-established in 2013. Now, it is possible for external requestors to search ES cells and vectors on the EuMMCR homepage (http://www.eummcr.org) and order them directly from the website. After finishing the order form, requestors receive an automatically created MTA in the PDF format by email. In order to finalize the order, the requestor prints the MTA, signs it and sends it to the legal department of the Helmholtz Zentrum München (HMGU).
Quality control: An important issue for WP10 is the quality control of the ES cell clones. Since January 2014, all the following quality controls are applied to every clone that the EuMMCR sends out. This is a huge improvement of the ES cell quality.
a. Mycoplasma PCR: all ES cells expanded and shipped by EuMMCR are tested for mycoplasma contamination using a PCR mycoplasma assay.
b. loxP PCR: the conditionality of the allele directly depends on the presence of both loxP sites flanking the critical exon. Due to recombination events within the area of the targeted region a loxP site can be absent in some of the EUCOMM alleles. The results of such recombination events are clones reflecting null mutation “without conditional potential”. Therefore EuMMCR is verifying the presence of both loxP sites, called “loxP PCR”, for all alleles.
c. genotyping: EuMMCR develops a genotyping protocol allowing the end user to genotype the ES cells and mutant mice easily. This genotyping LRPCR is performed with a primer in the targeting vector of the EUCOMMTOOLS allele and two gene specific primers outside the vector to confirm homologous recombination.
d. qPCR, copy number and chromosome check: In order to enable to exclude ES cell clones with double or multiple integrations of the targeting cassette, EuMMCR has established a neo and lacZ count qPCR. In addition, we check the chromosome numbers of Y, 8 and 11 to quickly exclude chromosomal alterations like trisomy 8 or 11, or loss of the Y chromosome.
WP 11 Training, Sustainability, Knowledge Dissemination and Outreach
WP Leader: W. Wurst (HMGU); Partners: all teams from WTSI, EMBL, TUD, CNR, GIE-CERBM, UMH and MRC
WP11 is concerned with the project’s training, sustainability, knowledge dissemination and outreach issues. To stringently manage and organize the EUCOMMTOOLS novel developments and production of conditional mutant materials monthly telephone conferences, meetings, and exchange with SAB have been organized. Face to face meetings of the scientists, steering committee and SAB took place regularly.
Monthly telephone conferences:
EUCOMMTOOLS organized regular internal consortium telephone conferences during the whole duration of the project to discuss project progress and problems. In general, these telephone conferences took place about once a month, with the exception of months where a progress meeting is organized, and of August (summer break). In addition, 10 consortium meetings took place to discuss progress, limitation, strategies, and novel developments to be integrated into the program. These meetings were also used for meetings of the steering committee and the scientific advisory board.
Consortium meetings:
- October 4-5, 2010: EUCOMMTOOLS kick-off meeting (Unterschleißheim, Germany)
- April 5-6, 2011: EUCOMMTOOLS Progress Meeting (Alicante, Spain)
- July 5-7, 2011: EUCOMMTOOLS Progress Meeting (Rome, Italy)
- April 3-4, 2012: EUCOMMTOOLS Progress Meeting (Hohenkammer, Germany)
- December 18-19, 2012: EUCOMMTOOLS Progress Meeting (Unterschleißheim, Germany)
- June 10-11, 2013: EUCOMMTOOLS Progress Meeting (Hinxton, UK)
- December 2-4, 2013: INFRAFRONTIER-IMPC-IKMC Meeting and EUCOMMTOOLS workshop (Rome, Italy)
- May 27-28, 2014: EUCOMMTOOLS Progress Meeting (Unterschleißheim, Germany)
- June 19, 2015: EUCOMMTOOLS Airport Meeting (Munich, Germany)
- September 3-4, 2015: EUCOMMTOOLS Farewell Meeting (Hohenkammer, Germany)
Steering Committee Meetings:
Meetings of the EUCOMMTOOLS Steering Committee (SC) took place in the framework of each of the EUCOMMTOOLS meetings mentioned above.
Scientific Advisory Board Meetings:
The EUCOMMTOOLS Scientific Advisory Board (SAB) consists of:
• Janan Eppig, The Jackson Laboratory, Bar Harbor, ME, USA
• John Hohmann, Allen Institute for Brain Science, Seattle, WA, USA (2011-2014)
• Alexandra Joyner, Memorial Sloan Kettering Cancer Center, New York, NY, USA
• Monica Justice, Houston, The Hospital for Sick Children (SickKids) in Toronto, Canada
• Björn Löwenadler, AstraZeneca R&D, Mölndal, Sweden
• Andrew McMahon, Harvard University, Cambridge, MA, USA
• Luis Puelles, University of Murcia, Spain
Three (J.E. B.L. and A.M.) of these advisors have already been members of the EUCOMM Scientific Advisory Board, so that they support continuity between the two projects.
The EUCOMMTOOLS SAB met once a year to give scientific advice to the project. According to this planning, the SAB was invited to the EUCOMMTOOLS progress meeting in Alicante, Spain (2011), the EUCOMMTOOLS progress meeting in Hohenkammer, Germany (2012), the EUCOMMTOOLS progress meeting in Hinxton, UK (2013), the EUCOMMTOOLS progress meeting in Unterschleißheim, Germany (2014), and the EUCOMMTOOLS farewell meeting in Hohenkammer, Germany (2015). Within these meetings the SAB discussed the project progress and developments with the EUCOMMTOOLS SC in separate sub-meetings and gave advice throughout the project.
Training courses:
Several training activities of ‘The Bioinformatics of Mutant Mouse Resources’ were performed by the EUCOMMTOOLS members EBI, WTSI and MRC Harwell. These training activities have been offered at MRC Harwell, UK on December 6-7, 2010 and, in addition, as a satellite workshop at the Mouse Genetics Meeting in Washington, DC, USA (June 22-25, 2011). The objectives of these two-day hands-on activities were, among others, to show participants how to find critical information about their favourite genes and to help them understand gene structures in detail as well as the underlying experimental data supporting the annotation. In addition, participants were enabled to find mutant mouse resources for their genes of interest using the IKMC and similar resources, to search IMPC phenotypic databases such as EuroPhenome and to gain an understanding of the high-throughput phenotype data generated and the statistical approaches required to analyse it.
Furthermore, a one day condensed version of the training was offered before the start of the Mouse Molecular Genetics Conference in Hinxton, UK (September 20, 2011) and was followed up with an afternoon session on mouse mutant resources during the 17:00-19:00 poster session on Sep. 21, 2011.
Responses to these training courses were very positive, and although originally not planned in Annex I, this training activity has continued during the reporting periods 2 and 3, having been offered at MRC Harwell, UK (June 18-19, 2012) and the European Bioinformatics Institute (EBI), UK (July 11, 2012), at Salamanca as part of a half-day workshop for the International Mammalian Genome Conference, Spain (September 15, 2013) and Hinxton, UK (September 19, 2013).
The advanced course “Genetic Manipulation of ES Cells” took place twice at the Wellcome Trust Sanger Institute Genome Campus in Hinxton, UK (October 31 - November 13, 2011; November 5 - November 18, 2012). This event was a two week intense practical and lecture course on gene targeting and genetic manipulation of mammalian stem cells, emphasizing mouse ES cells but including human iPS cells as well. Topics comprised gene annotation and KO design, vector construction through recombineering, ES cell culture, gene targeting, transposon technology, site-specific recombinases and RMCE, modular IKMC vector resources, genotyping and mouse production, homozygous ES cells, and TALEN assisted gene targeting. This course was originally not planned in Annex I but performed in addition.
The advanced course “Genetic Engineering of Mammalian Stem Cells”, which also took place at the Wellcome Trust Sanger Institute Genome Campus in Hinxton, UK (March 10th -22th, 2014), was a two week intense laboratory-based training course providing a comprehensive overview and practical laboratory experience of the genetic manipulation of mammalian stem cells. Focusing on mouse ES and human iPS cells, recent advances in genome informatics, recombineering, custom nucleases (CRISPRs/TALENs), transposon technology and conditional gene targeting were covered in lectures and through interactive demonstrations. Laboratory work included the culture and transfection of ES cells, design and construction of gene targeting vectors by BAC recombineering, and the use of PiggyBac transposons and designer nucleases (CRISPRs/TALENs) for advanced genome engineering. Participants were trained in the informatics and practical use of public gene targeting resources, including the production of mutant mice from the IKMC (International Knockout Mouse Consortium) resource. Emphasis was placed on the planning/design and successful experimental execution of gene targeting projects.
Two workshops were organized by Salvador Martinez, Almudena Martinez Ferre and Marusa Arencibia and took place at UMH Alicante, Spain. The workshop “Mouse Perfusion and Dissection” (May 7, 2013) aimed to instruct EUCOMMTOOLS work package 5.1 members in mouse perfusion and dissection, so that they are able to prepare samples for work package 6. The course was attended by Francesco Chiani and Alessia Gambadoro (CNR), Bruno Weber (ICS) and Ellen Brown, Nicola Griggs, Caroline Sinclair and Elizabeth Tuck (Sanger). Salvador Martinez (EUCOMMTOOLS work package 6 leader) introduced the course by describing the methodology and procedure for perfusion of P14 and P56 mice and visceral block extraction. Participants got to know the best fixation protocol for EUCOMMTOOLS samples, all suitable materials to be used and how to process the samples properly by immunohistochemistry. During the practical training, Salvador Martinez and Almudena Martinez showed how to perfuse P14 and P56 mice in situ and how to dissect the brain as well as thoracic and abdominal blocks of P56 mice. The second workshop “Tutorial on Mouse Anatomy and Gene Expression Patterns” (September 28-29, 2015) was designed to present the members the process of genetic annotation in relation to the project. The first part of the course had a theoretical approach in which knowledge were provided on the comparative anatomy of mouse and human body and the brain, being based on the ontology elaborated for the project. In the second part, the participants to the course applied all knowledge acquired in the theoretical sessions. Participants used the annotation platform that was designed for annotation. In particular, participants learned to use the annotation webpage and each of them annotated the expression of the gene reporter (Cre) of two samples of the project for both, the body and the brain. Moreover, they carried out a comparative expression analysis with other data bases. This was a very dynamic and interactive course and all participants acquired new knowledge on histology, and gene expression annotation.
Potential Impact:
Part IV: EUCOMMTOOLS Potential Impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results
Strategic impact
To be able to fight a disease efficiently, its cause(s) must be known. In particular, to understand the genetic contribution to disease etiology, genetic modified animal models are mandatory. These models are an absolute requirement to elucidate the function of (newly) identified disease genes, since knowing the function of a gene is essential for understanding the related disease processes. The physiological function of a gene can best be described via its loss-of-function in the whole organism. Thus the overall vision of the EUCOMM/EUCOMMTOOLS programs was to build the basis to the annotation of the function of all protein coding genes by producing mouse mutant resources for the world wide scientific community as basis to generate mouse models for elucidating gene functions.
In this regard EUCOMM/EUCOMMTOOLS are by now one of the key providers of mouse mutant resources worldwide and was a founding members of International Knockout Mouse Consortium (IKMC, http://www.mousephenotype.org) which provides the fundament of the International Mouse Phenotyping Consortium (IMPC, http://www.mousephenotype.org). Thus, together, in these international consortia null mutations of all protein coding genes are generated and their primary phenotypes are determined and an encyclopedia of gene function is currently generated. This encyclopedia will also form the reference for further more sophisticated disease models in which disease associated alterations will be recapitulated in vivo (i.e. introduction of disease associated mutations). All mutant embryonic stem (ES) cell and vector resources produced by EUCOMM/EUCOMMTOOLS are archived by the European Mouse Mutant Cell Repository (EuMMCR, http://www.eummcr.org) and all mutant mice at the European Mouse Mutant Archive (EMMA, http://www.infrafrontier.eu).
The success of EUCOMM/EUCOMMTOOLS programs is reflected by the fact that already more than 18.000 mutant ES cell have been produced by gene targeting and gene trapping by IKMC of which about 13.000 have been contributed by the EUCOMM/EUCOMMTOOLS consortia. It has to be noted that this responds to a coverage of the mouse genome of about 90%. The remaining 10% were not amenable to the EUCOMMTOOLS technology but can now be tracked by CRISPR/Cas9 technology. Proof of concept for large scale production has also been provided by EUCOMMTOOLS. More than 12.000 mutant ES cell clones and 1.000 gene targeting vectors have been distributed by EuMMCR to the scientific community upon request. Furthermore, based on this IKMC resource more than 5.000 mutant mice have been produced mostly by members of the IMPC and are currently phenotyped by IMPC and Infrafrontier and results are provided to the public. In addition, about 1.770 EUCOMM/EUCOMMTOOLS mouse lines have been archived by the EMMA project, and already about 1.489 of these mouse lines have been distributed to the international scientific community. The distribution of EUCOMM/EUCOMMTOOLS mice constitutes about 50% of the total EMMA/Infrafrontier distribution activities (pers. comm. Dr. Michael Hagn, EMMA project manager).
Concerning disease models the fact that for over 1.320 OMIM (Online Mendelian Inheritance in Man)-defined human genetic diseases at least one experimentally defined mouse model is available encompassing over 4.450 mouse models most of which are derived from IKMC, ECOMM/EUCOMMTOOLS mutant resource material (http://www.mousephenotype.org http://www.diseasemodels.org) (Eppig et al., NAR, 2015, de Angelis HM et al., 2015) also demonstrates the success of these programs and their absolute necessity for the scientific and medical community. In addition over the recent years it became apparent that many diseases present themselves as “syndromes”, that is, they present themselves with comorbide phenotypes. Thus the elucidation of gene function in specific organs and cell types, understanding the pleiotropic function of genes, is also crucial to understand disease etiology. To address this question conditional mutants are required which are provided by EUCOMM/EUCOMMTOOLS by the versatility of its products (“knock-out first, conditional ready”) since the loss-of-function mutant mice generated in the first instance can be simply converted to conditional mouse mutants by a single cross with another mouse line (i.e. the Cre-driver mouse line). Using these conditional mouse lines a gene can be inactivated in specific organs and cell types as well at specific time points by crossing them to appropriate Cre-driver lines, expressing a Cre-recombinase capable of inactivating the conditionally mutated gene in specific cell types and at specific time points during the lifespan of the mutant mouse, respectively.
In summary, the EUCOMM/EUCOMMTOOLS resources are heavily used by the scientific community as demonstrated by the distribution numbers of EuMMCR and EMMA/Infrafrontier and has substantially advanced the in vivo analysis of gene functions by IMPC/Infrafrontier and thus of the molecular genetic mechanisms underlying hereditary diseases (http://www.mousephenotype.org). Moreover, more than 1000 publications so far have acknowledged the use of EUCOMM/EUCOMMTOOLS material (google scholar) reflecting furthermore the success of these resources. Furthermore, the biotech company genOway is now distributing EUCOMM/EUCOMMTOOLS material to the pharmaceutical industry (see press release http://www.genoway.com/products-services/eucomm/eucomm-conditional-knockouts.htm). Thus, EUCOMM/EUCOMMTOOLS products are also of commercial value and have impact on European competitiveness in biotech and pharmaceutical industry.
European approach
The knowledge, technological expertise and experience as well as infrastructure brought together for the EUCOMMTOOLS proposal can certainly not be found in a single institution or country; therefore, this consortium has been built on a Pan-European level. In addition, no single country could afford the finances for a project of this dimension, thus, EU funding has been necessary to implement this undertaking. Europe has already taken the lead in a number of new research approaches that are fundamental for the future use of the mouse as a central model organism (e.g. the EU programmes EUCOMM/EUCOMMTOOLS, I-DCC, CREATE, EUMODIC, Infrafrontier). Among these projects, certainly, the EUCOMM/EUCOMMTOOLS program laid the groundwork for Europe’s leadership in high-throughput gene function annotation. The mission of the EUCOMM/EUCOMMTOOLS project has been, as detailed below, to provide sophisticated tools enabling researchers world-wide to capitalize on the EUCOMM/EUCOMMTOOLS and IKMC resources, as these resources will allow an unprecedented fine-tuning of disease analysis for about 90% of all protein coding genes. Thus, EUCOMMTOOLS was instrumental in consolidating and even extending Europe’s leadership in large scale and mouse functional genomics. In particular, it also lays the ground, know-how and tools to functionally validate the outcome gathered in systems biology research approaches in vivo. In addition, the EUCOMMTOOLS vector toolkit allows introducing various human disease associated alleles for mimic human diseases, thus, EUCOMMTOOLS will build a bridge between research using cutting-edge mouse models and approaches using human disease associated alleles. Furthermore, all vector tools produced by EUCOMMTOOLS can readily be used to study disease mechanisms in human ES and iPS cells.
The EUCOMMTOOLS project is highly integrated with other national, European and international initiatives:
1) In the EUCOMMTOOLS work packages knowledge and results generated by several other EU programs are directly included, as the CREATE program in work packages 1 and 9, EURExpress in work package 1 and, as the Cre expression analysis is done by an expert also involved in EURExpress gene expression annotation, also in work package 6, as well as EUMODIC in work package 7, as the EUMODIC coordinator will perform mouse phenotyping in this work package according to the EUMODIC standards. In addition, a national United Kingdom initiative will be integrated into EUCOMMTOOLS, namely the establishment of the knockout miRNA resource “mirKO” at the Wellcome Trust Sanger Institute which is supported by institutional Wellcome Trust funds.
2) The I-DCC consortium has been a close EUCOMMTOOLS collaboration partner and integrated all mouse genomics and functional genomics data in one IKMC web portal (http://www.knockoutmouse.org). Furthermore, the EUCOMMTOOLS consortium tightly cooperates with the EMMA/Infrafrontier consortium, which distributes the Cre driver and conditional knockout mice produced by EUCOMMTOOLS and the scientific community. On the international level, EUCOMM/EUCOMMTOOLS contributed certainly most resources to IKMC, which is composed in addition of KOMP, NorCOMM, and TIGM. In addition, a NorCOMM successor proposal (NorCOMM2) which is generating Cre driver lines mostly expressed in stem cell populations using a transposon approach (coordinator: Colin McKerlie). This undertaking is complementing the EUCOMMTOOLS approach. EUCOMMTOOLS tightly cooperates with NorCOMM2 to prevent redundancies. Furthermore, the group of Prof. Simpson at the University of British Columbia coordinated the CanEuCre proposal in close cooperation with EUCOMMTOOLS. The goals of this proposal were to establish 30 Cre driver transgenic lines using predetermined mini and maxi promoters and a knock-in approach in the Hprt locus. Furthermore, 25 AAV virus containing Cre vectors have been generated for direct application in vivo. CanEuCre is integrated into, and its materials registered and distributed by, the IKMC. CanEuCre was a close collaboration partner of EUCOMMTOOLS, also complementing its Cre driver approach.
3) Via its work package 9 ‘International coordination and databases’ EUCOMMTOOLS is integrated into the IKMC/IMPC database. This WP also coordinated international effort to harmonize SOP, to prevent overlaps and redundancy to generate synergies to create a unique resource under the umbrella of IKMC and IMPC.
In summary, the EUCOMM/EUCOMMTOOLS program is an extremely well integrated initiative which is part of a singular international undertaking representing the world’s largest collection of, predominantly conditional, mutant murine materials (vectors, vector toolkits, ES cells, Cre driver and knockout mouse models), easily searchable via the common IKMC web page (www.mousephenotype.org) which is connected to the various IKMC members’ databases and which material is readily available and distributed via EuMMCR, MMCR and EMMA/Infrafrontier to academic and commercial customers worldwide.
Dissemination of Project Results
Dissemination of Knowledge
In general, the scientific results of EUCOMMTOOLS have been disseminated to the scientific community by the classical means of publication in peer review scientific journals and by presentation at scientific conferences and webportals. So far, 167 manuscripts of EUCOMMTOOLS members have been published related to the EUCOMMTOOLS project. Moreover, already more than 1000 publications acknowledged the use of EUCOMM/EUCOMMTOOLS material (google scholar, December 2015).
In addition, we introduced the EUCOMMTOOLS project to the scientific community by producing flyers and posters. Furthermore, as known from the EUCOMM program, a resource producing project’s home page has been established (http://www.eucommtools.org which directs the user to the EUCOMMTOOLS project website, now embedded into IMPC website www.mousephenotype.org ). As EUCOMMTOOLS builds on and enhances the EUCOMM resource, intentionally the EUCOMMTOOLS name and logo have received the characteristics of a EUCOMM name and logo add-on, and a common EUCOMM/EUCOMMTOOLS/IKMC/IMPC home page was established which is now being integrated into the IMPC web portal. The latter is particularly important as EUCOMM is already well-known, as also stated by the EUCOMM reviewers, in the wider scientific community. Thus, EUCOMM has acquired ‘brand’ characteristics which should be extendable to EUCOMMTOOLS via public relations measures. In addition, the EUCOMM/EUCOMMTOOLS home page is approachable via the IKMC home page, and EUCOMMTOOLS’ data integrated into the IKMC searchable database. On the EUCOMM/EUCOMMTOOLS home page also all protocols for handling of the consortia’s materials will be found, e.g. for electroporation, cell culture, microinjection etc.
EUCOMMTOOLS’ knowledge will also be disseminated via the other EU projects directly integrated into the consortium and by networking with different national, European, and international projects, as e.g. several projects funded within the eMed Systems Medicine Research Network, Infrafrontier, and the International Mouse Phenotyping Consortium (IMPC).
EUCOMMTOOLS’ work package ‘International coordination and databases’ also contributes substantially to international collaboration in collating information on the activities in Europe and worldwide in the field of conditional mouse mutant technologies, determining the degree of integration and discussing ways to improve coordination. All international activities have been coordinated with our IKMC partners.
Dissemination of EUCOMMTOOLS materials
In addition to knowledge, the materials produced by EUCOMMTOOLS are disseminated to the scientific community by the EuMMCR (vectors, toolkits, ES cells) and by the EMMA/Infrafrontier consortium (mice).
The EuMMCR (http://www.eummcr.org) evolved in the framework of the EUCOMM project as EUCOMM’s material distribution unit, but also archives and distributes material of other EU funded projects such as EuTRACC and SYBOSS materials. The EuMMCR became part of the Helmholtz Zentrum München in November 2007 and, since then, predominantly distributes quality controlled mutant ES cells (more than 12.000 clones to date), but also gene targeting (more than 1000 to date) and gene trapping vectors to the academic scientific community world-wide. EUCOMM/EUCOMMTOOLS material is now also distributed to commercial partners via genOway worldwide (see press release at the EuMMCR homepage, www.eummcr.org). EuMMCR’s customers are predominantly located in Europe (51%) and the US/Canada (40%), and 9% in other countries. In addition, the EuMMCR also expanded, quality controlled and shipped another about 2.500 clones to IMPC members for mouse production. The EuMMCR is a very successful distribution unit with proven qualification to distribute EUCOMMTOOLS vectors, toolkits and mutant ES cells under quality controlled SOPs efficiently (see also work package 10).
The distribution of EUCOMM/EUCOMMTOOLS mice is performed by EMMA/Infrafrontier. So far, more than 2.000 EUCOMM/EUCOMMTOOLS derived mouse strains have been archived, more than 1.700 strains have been ordered and more than 1.500 have been distributed. In addition, mice produced by The Jackson Laboratory will be distributed by this institution. All EUCOMM and EUCOMMTOOLS materials are/will be distributed to the academic and industrial scientific, and thereby biomedical, community. Even after the end of the funding of EUCOMM/EUCOMMTOOLS all material will be maintained and distributed to the scientific community by a long term self-sustaining concept providing the material to the customer at cost prize. Thus, EUCOMM/EUCOMMTOOLS has established a long term self-sustainable concept far beyond direct funding of the EU.
Commercial exploitation of project results, and plan for management of knowledge (Intellectual Property)
Commercial exploitation issues are taken care of by the Wellcome Trust Technology Transfer Division (WTTTD) together with Ascenion GmbH as counselors of the management team and the steering committee as well as by the Technology Transfer Units of all EUCOMMTOOLS participants. These units have settled a deal with genOway to distribute EUCOMM/EUCOMMTOOLS material to commercial end users. The EUCOMMTOOLS conditional knockout resources are now also available to commercial entities.
In principle, all EUCOMMTOOLS products are of commercial interest, i.e. gene targeting vectors, gene targeting vector design software, conditionally targeted mouse ES cells, conditionally targeted mice, Cre driver mice, as well as products of the EUCOMMTOOLS technology development work package, namely recombinases, new gene cassettes, new recombinase inducible systems etc. For example, conditionally mutated ES cells, and mice derived from such ES cells, are of interest for companies performing pharmaceutical research and development. Besides the elucidation of gene functions, ES cell clones can be used by biotech and pharma companies for preclinical research and development, typically in the so-called ADME&Tox applications (evaluation of the adsorption, distribution, metabolism, excretion and toxicology profile of a compound of interest). Furthermore, these models are of extreme value for drug target validation to determine potential side effects. Moreover, we seek intellectual property protection whenever possible supported by WTTTD and Ascenion GmbH.
As stated above, we have established a commercial distribution via the SME genOway (France). For more detailed information please follow the link to the press release “genOway offers industry scientists immediate access to EUCOMM conditional KO mouse models” which can be found at: https://www.eummcr.org/ .
EUCOMMTOOLS distributes its resources to as many for-profit entities as possible to maximize the commercial as well as the scientific impact of the resources. EUCOMMTOOLS does not entertain exclusive arrangements with any commercial entity for access to the project’s technology developments or resources. In general, the EuMMCR distributes vectors and ES cells to industry via genOway, and mice will be distributed to industry by EMMA/Infrafrontier or the consortium participant who has generated the mice via genOway as well. The revenues for the commercially distributed materials have to be divided between the EUCOMMTOOLS members. In addition, the EuMMCR, when involved, and the technology transfer unit shall receive remuneration. An adequate split scheme is defined in the project’s consortium agreement.
Finally, the commercial exploitation of EUCOMMTOOLS materials has also got high socio-economic impact as e.g. the use of mutant ES cells and mice for pre-clinical research and development will allow an enhancement of diagnostics and drug development, and thereby a more readily available treatment of citizens, reducing unnecessary and expensive medical approaches.
In summary, the EUCOMMTOOLS scientific, societal, and economic impact increased by manifold, tailor-made models for drug screening, validation is made available and to enhance knowledge of gene function and their contributions to health and disease.
List of Websites:
Scientific project coordinator: Prof. Wolfgang Wurst, Helmholtz Zentrum Munich, Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Tel: +49 89 3187 4110
Fax: +49 89 3187 3099
Email: wurst@helmhotz-muenchen.de
Project website: http://www.mousephenotype.org