EUCOMMçthe European Conditional Mouse Mutagenesis Program Roland H. Friedel, Claudia Seisenberger, Cornelia Kaloff and Wolfgang Wurst

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1 BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS. VOL 6. NO ^185 doi: /bfgp/elm022 EUCOMMçthe European Conditional Mouse Mutagenesis Program Roland H. Friedel, Claudia Seisenberger, Cornelia Kaloff and Wolfgang Wurst Advance Access publication date 29 October 2007 Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource. Keywords: mouse mutagenesis; gene trap; gene targeting; conditional allele INTRODUCTION The recent advancements in high-throughput DNA sequencing have generated a series of complete genome sequences including those of mouse and man. The annotation of these genomes has resulted in a comprehensive directory of the genes that make up a living organism. After this initial phase of gene discovery, biomedical research is now faced with the challenge to elucidate the functions of individual genes in the context of the entire organism. Functional genome annotation will ultimately lead to comprehension of the genetic basis of human health and various disease conditions. The mouse is ideally suited for the functional analysis of the genome for several reasons: first, the mouse genome shows a high degree of homology with the human genome; second, the mouse has an excellent track record as a model system for human traits and diseases and finally, an arsenal of molecular and embryologic technologies makes it possible to easily mutate genes in mouse embryonic stem (ES) cells and to generate mice that carry defined mutations. The European Conditional Mouse Mutagenesis Program (EUCOMM) takes the lead in saturation mutagenesis of the mouse genome, which will be the basis for functional annotation of the entire genome [1, 2]. This review describes the structure and the goals of the EUCOMM program as well as the technologies that have been implemented to generate this resource. THE EUCOMM PROGRAM EUCOMM is a collaborative effort of 9 participants from four European countries (Figure 1), coordinated by Wolfgang Wurst from the GSF-National Research Center for Environment and Health, Munich-Neuherberg, Germany and Allan Bradley from the Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. The EUCOMM program is funded by the European Union as part of Framework Program 6. Mutagenesis in EUCOMM will be performed in a high throughput manner using conditional gene trapping and conditional gene targeting strategies. Corresponding author. Wolfgang Wurst, GSF National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, Neuherberg, Germany. Tel: þ ; Fax: þ ; wurst@gsf.de Roland Friedel is team leader of the EUCOMM team at the Institute of Developmental Genetics. Claudia Seisenberger is team leader of the gene targeting group at the Institute of Developmental Genetics. Cornelia Kaloff is project manager of the EUCOMM program. WolfgangWurst holds the Chair of Developmental Genetics at the Technical University of Munich and is director of the Institute of Developmental Genetics at the GSF National Research Center. He is coordinator of the EUCOMM program. ß Oxford University Press, 2007, All rights reserved. For permissions, please journals.permissions@oxfordjournals.org

2 EUCOMM 181 Gene Trapping Gene Targeting Mouse production Distribution Vector development University of Frankfurt, D ES cell clone production Enabling technologies U. of Technology Dresden, D Vector development Generation of Cre ER T2 mice ICS Strasbourg, F EMBL Monterotondo, I Distribution of mutant ES cells and vectors EUCOMM Distribution Center PCR of gene trap insertions Charité Berlin, D Sequencing and annotation ES cell clone production ES cell clone validation Mutant mouse production CNR-IBC Monterotondo, I ICS Strasbourg, F MRC MGU Harwell, UK Archiving and distribution of mice The European Mouse Mutant Archive (EMMA) Database Figure 1: Organizational structure of the EUCOMM program. EUCOMM comprises 9 research institutions from four European countries (see text for affiliations of EUCOMM partners). The tasks performed by these institutions are shown here. The conditional mutagenesis approach allows the analysis of gene functions in a time- and tissuespecific manner. This feature is especially important for about 20% of the genes whose non-conditional mutation causes embryonic lethality, thus precluding analysis at later stages [3, 4]. Furthermore, conditional mutagenesis will help to create better mouse models for those human disease conditions which are caused by somatic mutations in the adult patient. Thus, EUCOMM generates a unique resource that will, for the first time, permit to analyse gene function on a large scale. The EUCOMM program will mutate a substantial fraction of the mouse genome. It is planned to produce up to gene trap mutations and gene targeting mutations in mouse ES cells. To facilitate the initial analysis of these mutations, EUCOMM will also generate up to 320 mutant mice from its resource. Furthermore, to increase the tools for conditional mutagenesis, EUCOMM intends to establish 20 ligand-inducible Cre driver lines with novel tissue-specific expression patterns. All EUCOMM reagents (plasmids, ES cells and mice) will form an open resource for the international scientific community. Further details about the EUCOMM program can be found on the EUCOMM website It should be noted that this webpage also provides access to the list of genes chosen for conditional gene targeting in EUCOMM, and scientists are invited to prioritize their genes of interest on this list (procedure described on EUCOMM website). EUCOMM GENE TRAPPING The gene trapping strategy relies on the random insertion of a gene trap vector into an intron of a transcribed gene [5]. The insertion of the vector disrupts the function of the trapped gene and simultaneously reports its expression. The main advantage of gene trapping is that a single vector can be used to mutate a large number of genes in a limited number of experiments. Gene trapping has been successfully applied to create a large collection of mutant mouse ES cells over the last 10 years (see the website of the International Gene Trap Consortium, [6]). Based on previous gene trapping efforts, it is estimated that gene trapping can cover up to 60% of all mouse genes [7, 8]. The EUCOMM gene trap vector is based on the FlipROSAbgeo vector, which is delivered to ES cells as a moloney murine leukemia virus (MMLV) retrovirus (Figure 2A). The promotorless trap cassette

3 182 Friedel et al. A B C D E F Gene trap vector FRT F3 loxp lox5171 Trapped allele (invertible) Conditional allele Trapped allele (reinverted) LTR LTR + FLP recombinase: Inversion + FLP recombinase: Excision + Cre recombinase: Inversion + Cre recombinase: Excision Figure 2: Gene inactivation with the EUCOMM gene trap vector. (A) Schematic representation of the retroviral gene trap vector rsfliprosabgeo. LTR, long terminal repeat; FRT and F3, heterotypic target sites for FLP recombinase; loxp and lox5171, heterotypic target sites for Cre recombinase; SA, splice acceptor; bgeo, b-galactosidase/neomycinphosphotransferase fusion gene; pa, polyadenylation sequence. (B) Schemeofa trapped allele [shown here, as an example, is an insertion between exon 1 () and exon 2 ()]. The integration of the gene trap vector leads to a splicing of exon1to the splice acceptor of the vector, thereby disrupting gene function and simultaneously reporting the expression pattern of the trapped gene. (C) Transient step during the generation of a conditional gene trap allele. The FLP recombinase can invert the vector cassette by recombination of either the FRT or F3 sites (shown here is an inversion via the FRT sites, indicated by curved arrows). (D) Conditional allele. The inversion through the FRT sites has placed the F3 sites in tandem orientation. The recombination of the F3 sites deletes one FRT and onef3site(indicatedbycurvedarrows),therebypreventing further FLP activity.this allele is non-mutagenic, because the splice acceptor of the trap vector is facing away from the direction of transcription. (E) Transient step during the reinversion of the trap vector. The Cre recombinase can invert the vector cassette by recombination of either the loxp or lox5171sites (shown here is an inversion via the loxp sites, indicated by curved arrows). (F) Reinverted trapped allele. The inversion through the loxp sites has placed the lox5171 sites in tandem orientation. The recombination of the lox5171 sites (indicated by curved arrows) deletes one loxp and one lox5171 site, thereby preventing further Cre activity. This allele is mutagenic, because the splice acceptor is in the direction of transcription. Figure modified after [10]. consists of a splice acceptor, a b-gal reporter, a neomycin resistance gene and a polyadenylation signal. To improve the trapping rates for genes encoding secreted or transmembrane proteins, a variant of the vector, rsfliprosaceo, which contains an internal transmembrane domain, will be used in parallel [9]. To achieve the conditionality of the trap alleles, the gene trap vector utilizes the FlEx technology developed by F. Schnütgen [10]. Two pairs of heterotypic recombination sites for the Cre and FLP recombinases, respectively, are positioned in inverted orientation around the trap cassette. This allows inverting the cassette two times. The first inversion silences the mutation and creates a conditional allele (Figure 2B D). The second inversion reinstates the mutation (Figure 2E and F). The first inversion can be either done in vitro by transiently transfecting a trapped ES cell line with, for example, FLP recombinase, or invivo by breeding a gene trap mouse line with a mouse line that expresses FLP in its germline ( FLP deleter mouse ). A mouse carrying such an inverted, i.e. conditional, allele can then be bred to Cre driver lines. Depending on the expression properties of the respective Cre line, the mutation can be selectively reactivated in a time- and tissue-specific manner. Alternatively, Cre recombinase can be introduced into a defined tissue location by viral delivery. Gene trapping is a random mutagenesis approach; consequently, the rate of newly trapped genes will decline continuously during the course of trapping [3]. When the trapping rate of new genes is no longer cost efficient, the EUCOMM gene trapping efforts will switch completely to gene targeting. EUCOMM GENE TARGETING Gene targeting, also known as Knock-out, relies on homologous recombination to introduce a DNA vector into a chosen locus [11]. The EUCOMM targeting vectors will be mainly designed as promoterless targeting vectors that contain a splice acceptor ( targeted trapping ; [12]). The promoterless design makes such vectors more selective for correct recombination events, thus increasing the targeting frequencies and reducing the effort necessary for obtaining targeted ES cell clones. Similar to gene trapping, the targeted trapping strategy can only hit genes that are expressed in ES cells. Because of that, the first set of EUCOMM

4 EUCOMM 183 A Targeting vector 5 homology arm FRT loxp critical exon 3 homology arm B Targeted allele + FLP recombinase OR + Cre recombinase C E Conditional allele + Cre recombinase lacz-tagged null allele (D exon) D Null allele (D exon, frameshift) Figure 3: Gene inactivation with the EUCOMM gene targeting vector. (A) Schematic representation of a targeted trapping vector.two homology arms at the 5 0 and 3 0 ends of the vector mediate gene specific targeting by homologous recombination. A SA^b geo ^ pa cassette allows promoterless targeting ( targeted trapping ). A critical exon (that is, an exon whose excision creates a frame shift) is flankedby loxp sites [here, as an example, exon 2 ()].FRT, target site for FLP recombinase; loxp, target site for Cre recombinase; SA, splice acceptor; bgeo, b-galactosidase/neomycinphosphotransferase fusion gene; pa, polyadenylation sequence. (B) Targeted allele. In this example, the targeting strategy leads to the insertion of the SA^b geo ^pa cassette into the intron between exons 1 and 2. This cassette acts like a gene trap insertion and disrupts gene function and reports the expression of the targeted gene. (C) Conditional allele after excision of the SA^b geo ^pa cassette by FLP recombinase.gene function is restored. (D) Null allele after excision of the critical exon by Cre recombinase. (E) As an alternative to the subsequent recombination steps in (C) and (D), recombination of the targeted allele with Cre recombinase leads to a null allele that reports gene expression. Figure modified after [16]. target genes is assembled from genes that are present at least once in a public or private gene trap resource (indicating that these genes are amenable to promoterless targeting). Genes that are not expressed in ES cells will be targeted with a vector that contains a neomycin resistance gene driven by a phosphoglycerine kinase (PGK) promoter. The conditional gene targeting vectors are generated from a bacterial artificial chromosome (BAC) library of mouse genomic DNA by recombineering technology [13]. The EUCOMM targeting vector was developed by W. Skarnes and B. Rosen at the Sanger Institute based on a strategy described by F. Stewart and colleagues [14]. The vector contains a targeting cassette with a splice acceptor followed by a b-gal reporter and a neomycin resistance gene that disrupt gene function and report gene expression in a similar manner to the gene trap vector. The targeting cassette is flanked by FRT sites, so that it can be removed by FLP recombinase. In addition, the vector contains two loxp sites that flank a critical exon (Figure 3A). The deletion of this critical exon by Cre recombinase causes a frame shift between the remaining exons, and thus the complete disruption of gene function. To convert a targeted allele into a conditional allele, the targeting cassette has to be removed first by FLP recombination (Figure 3C). In a mouse that carries such a conditional allele, gene activity can then be selectively inactivated by Cre recombinase, which can be introduced either as transgene or by viral delivery (Figure 3D). If a conditional allele is not desired, it is also possible to use Cre recombinase directly on the original allele to create a lacz-tagged null allele (Figure 3E). EUCOMM CRE LINES To complement the library of conditional alleles, the EUCOMM program will also generate up to 20 new mouse lines that will express ligand-inducible Cre

5 184 Friedel et al. recombinase in a tissue-specific manner. Amongst these lines will be mice that express Cre, for example, in certain subsets of muscle or heart cells, subsets of pancreatic cells or subsets of neurons such as adult neuronal stem cells, serotonergic or dopaminergic neurons. These lines will be made either by pronuclear injections of BACs that contain particular promoter regions of genes together with a cassette encoding Cre, or by recombinase-mediated cassette exchange in EUCOMM ES cells (replacing the trapping/targeting cassette with a Cre cassette). The version of Cre enzyme that will be used for EUCOMM will be one that is inducible by tamoxifen (Cre-ER T2 ), which enables temporal control of Cre activation [15]. THE EUCOMM RESOURCE The EUCOMM ES cell lines will be distributed centrally by the EUCOMM Distribution Center at the GSF Research Center in Munich-Neuherberg. Access to the database of EUCOMM genes can be found under The EUCOMM Distribution Center will also send out the intermediate and final plasmid vectors used for EUCOMM gene targeting. It is noteworthy that these plasmids can be easily modified by users to generate knock-outs of their own design. The mice generated in the EUCOMM project will be archived as frozen embryos or sperm for long-term storage. The European Mouse Mutant Archive network (EMMA; will disseminate the mutant mice to the scientific community upon request. THE INTERNATIONAL MOUSE KNOCKOUT CONSORTIUM To make the functional annotation of the entire mouse genome achievable over the next few years, it had been recognized by leading biomedical scientists that it is essential to coordinate the different international mouse mutagenesis programs [1, 2]. The cornerstones of this worldwide mutagenesis effort are the European EUCOMM program, the US KnockOut Mouse Project (KOMP) and the Canadian North American Conditional Mouse Mutagenesis Project (NorCOMM), which together form the International Mouse Knockout Consortium (IMKC; [16]). To this international initiative, EUCOMM plans to contribute the largest fraction of mutant mouse ES cell lines. Furthermore, EUCOMM put forward the use of conditional mutagenesis, an approach which has been adopted by most IMKC partners. It is planned to exchange the EUCOMM, KOMP and NorCOMM resources to establish a unique, comprehensive resource useful for scientists worldwide. EUCOMM PARTNERS GSF-National Research Center for Environment and Health, GmbH, Munich/Neuherberg, Germany [Prof. Wolfgang Wurst, (EUCOMM coordinator), Prof. Martin Hrabé de Angelis, Dr Andreas Hörlein], the Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK [Prof. Allan Bradley, (EUCOMM coordinator), Dr William Skarnes, Dr Pentao Liu, Dr Anthony Cox], the University of Frankfurt Medical School, Frankfurt/Main, Germany (Prof. Harald von Melchner), the Charité Universitätsmedizin, Berlin, Germany (Prof. Patricia Ruiz), the University of Technology Dresden, Dresden, Germany (Prof. Francis Stewart), the Institut Clinique de la Souris (ICS), Strasbourg, France (Prof. Johan Auwerx/Prof. Pierre Chambon), the European Molecular Biology Laboratory (EMBL), Monterotondo, Italy (Prof. Nadia Rosenthal), the Medical Research Council (MRC), Mammalian Genetics Unit, Harwell, UK (Prof. Steve Brown), the Consiglio Nazionale delle Ricerche (CNR), IBC, Monterotondo, Italy (Prof. Glauco Tocchini-Valentini) European Commission Project Scientific Management (Christian Desaintes, Jacques Remacle Octavi Quintana-Trias). Key Points The European Conditional Mouse Mutagenesis Program (EUCOMM) aims at mutating up to genesby gene trapping and up to 8000 genes by gene targeting in mouse embryonic stem (ES) cells. The mutations can be rendered conditional, so that each mutation can be activated by Cre recombinase in a time- and tissue-specific manner. EUCOMM will generate up to 320 mouse lines from its mutant ES cells. EUCOMM will generate up to 20 mouse lines expressing Cre in a tissue-specific manner. EUCOMM will distribute its resource to the scientific community. The program is part of an international effort to generate a resource of mutations for all mouse genes.

6 EUCOMM 185 Acknowledgements We wish to thank all members of the EUCOMM program for fruitful discussions and for their collegial spirit in this collaborative effort. We especially thank A. Bradley, W. Skarnes and J. Auwerx for sharing targeting vectors and cell lines with the EUCOMM team at the GSF National Research Center. References 1. Austin CP, Battey JF, Bradley A, etal. The knockout mouse project. Nat Genet 2004;36: Auwerx J, Avner P, Baldock R, et al. The European dimension for the mouse genome mutagenesis program. Nat Genet 2004;36: Hansen J, Floss T, Van Sloun P, et al. A large-scale, genedriven mutagenesis approach for the functional analysis of the mouse genome. Proc Natl Acad Sci USA 2003;100: Mitchell KJ, Pinson KI, Kelly OG, et al. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat Genet 2001;28: Stanford WL, Cohn JB, Cordes SP. Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet 2001;2: Nord AS, Chang PJ, Conklin BR, et al. The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse. Nucleic Acids Res 2006;34:D Skarnes WC, von Melchner H, Wurst W, et al. A public gene trap resource for mouse functional genomics. Nat Genet 2004;36: Zambrowicz BP, Abuin A, Ramirez-Solis R, et al. Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. Proc Natl Acad Sci USA 2003;100: De-Zolt S, Schnutgen F, Seisenberger C, et al. Highthroughput trapping of secretory pathway genes in mouse embryonic stem cells. Nucleic Acids Res 2006;34:e Schnütgen F, De-Zolt S, Van Sloun P, et al. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc Natl Acad Sci USA 2005;102: Capecchi MR. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 2005;6: Friedel RH, Plump A, Lu X, et al. Gene targeting using a promoterless gene trap vector ( targeted trapping ) is an efficient method to mutate a large fraction of genes. Proc Natl Acad Sci USA 2005;102: Testa G, Zhang Y, Vintersten K, et al. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol 2003;21: Testa G, Schaft J, van der Hoeven F, et al. A reliable lacz expression reporter cassette for multipurpose, knockout-first alleles. Genesis 2004;38: Indra AK, Warot X, Brocard J, et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifeninducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 1999;27: Collins FS, Rossant J, Wurst W. A mouse for all reasons. Cell 2007;128:9 13.