Mamm Genome DOI 10.1007/s00335-013-9467-x Molecular Characterization of Mutant Mouse Strains Generated from the EUCOMM/KOMP-CSD ES Cell Resource Edward Ryder • Diane Gleeson • Debarati Sethi • Sapna Vyas • Evelina Miklejewska • Priya Dalvi • Bishoy Habib • Ross Cook • Matthew Hardy • Kalpesh Jhaveri • Joanna Bottomley • Hannah Wardle-Jones • James N. Bussell • Richard Houghton • Jennifer Salisbury • William C. Skarnes • Sanger Mouse Genetics Project • Ramiro Ramirez-Solis Received: 3 April 2013 / Accepted: 27 June 2013 Ó The Author(s) 2013. This article is published with open access at Springerlink.com Abstract The Sanger Mouse Genetics Project generates Introduction knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the The extensive genetic resources available for the mouse, consequences of the mutations using a high-throughput including the sequencing and annotation of the genomes of primary phenotyping screen. Upon achieving germline multiple inbred laboratory strains (Church et al. 2009; transmission, new strains are subject to a panel of quality Keane et al. 2011; Flicek et al. 2012; Wong et al. 2012), control (QC) PCR- and qPCR-based assays to confirm the have facilitated comprehensive comparisons with the correct targeting, cassette structure, and the presence of the human genome (Guigo et al. 2003; Zheng-Bradley et al. 30 LoxP site (required for the potential conditionality of the 2010; Mouse ENCODE Consortium et al. 2012). This allele). We report that over 86 % of the 731 strains studied makes the mouse a powerful tool for both investigating showed the correct targeting and cassette structure, of gene function and modelling disease progression in mam- which 97 % retained the 30 LoxP site. We discuss the malian systems. This importance can be demonstrated by characteristics of the lines that failed QC and postulate that the wealth of resources available for researchers studying the majority of these may be due to mixed ES cell popu- human diseases and genetic disorders, including (but not lations which were not detectable with the original limited to) cancer (Frese and Tuveson 2007; Kim and Baek screening techniques employed when creating the ES cell 2010; Leystra et al. 2012), visual (Gao et al. 2002; van de resource. Pavert et al. 2007) and auditory dysfunctions (Leibovici et al. 2008; Spiden et al. 2008), neurodegenerative condi- tions (Games et al. 1995; Schilling 1999; Ravikumar et al. 2004; Wirths and Bayer 2010), and diabetes (Cho et al. 2001; Duan et al. 2004). There are over 1,100 human diseases with one or more mouse models, and over 3,600 mouse genotypes model human disease as reported at the Electronic supplementary material The online version of this article (doi:10.1007/s00335-013-9467-x) contains supplementary Mouse Genome Database (MGD) (http://www.informatics. material, which is available to authorized users. jax.org, December 2012). To facilitate these investigations, several large-scale efforts to create knockout mutations in & E. Ryder ( ) Á D. Gleeson Á D. Sethi Á S. Vyas Á mice have been established (Bradley et al. 2012) by the E. Miklejewska Á P. Dalvi Á B. Habib Á R. Cook Á M. Hardy Á K. Jhaveri Á J. Bottomley Á H. Wardle-Jones Á systematic construction of targeted mutations (Valenzuela J. N. Bussell Á R. Houghton Á J. Salisbury Á et al. 2003; Prosser et al. 2011; Skarnes et al. 2011). W. C. Skarnes Á Sanger Mouse Genetics Project Currently, the largest resource of targeted mutations is the & R. Ramirez-Solis ( ) EUCOMM/KOMP-CSD mouse embryonic stem cell (ESC) The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK collection (Skarnes et al. 2011), which is based on JM8 e-mail: [email protected] agouti or non-agouti C57BL/6N ES cells (Pettitt et al. R. Ramirez-Solis 2009). The structure and modification of the promoter- e-mail: [email protected] driven ‘‘knockout-first’’ EUCOMM/KOMP-CSD allele, 123 E. Ryder et al.: EUCOMM/KOMP-CSD Mouse QC which forms the majority of the collection, is shown in genes, 56 % of the 22,147 CCDS (Pruitt et al. 2009) gene Fig. 1. models present in Ensembl (Flicek et al. 2012). The The EUCOMM/KOMP-CSD collection, along with resource was generated by use of a high-throughput mod- those generated by Regeneron, and the Canadian Nor- ular gateway-based vector construction and positive–neg- Comm programme form the International Mouse Knockout ative selection for high-efficiency targeting in ES cells Consortium (IKMC) resource (Collins et al. 2007; Ring- (Skarnes et al. 2011). Clones were then screened by long- wald et al. 2011; Bradley et al. 2012) and are the main range PCR and sequencing to confirm targeting and the source of ES cells used for mouse production by the presence of the 30 loxP site that is required for the condi- International Mouse Phenotyping Consortium (IMPC) tionality of the mutant allele. Although this approach is (Brown and Moore 2012). appropriate for a high-throughput pipeline in terms of cost The goal of the IMPC is to generate knockout strains for and speed, it does have its limitations. For example, long- all protein-coding genes in the mouse on a pure C57BL/6N range PCR is likely to miss mutations within the cassette genetic background, and to elucidate gene function by use and is not able to detect mixed ESC populations. As the of a broad-spectrum high-throughput primary phenotyping resource is exploited to generate mouse lines, it will be screen. These phenotypes can then be studied in more important to ascertain the molecular structure of the alleles depth by the scientific community at large within special- transmitted to mice. ized areas of interest. Here we present a detailed and extensive molecular The aims of the IMPC overlap with the Wellcome Trust characterization of the mutant alleles in mouse strains Sanger Mouse Genetics Project (Sanger MGP) (White et al. generated from the resource. We demonstrate that although 2013) which was formed in 2006 to generate and phenotype the majority of the mouse lines produced by Sanger MGP 200 mutant mouse strains per year using a battery of tests from the EUCOMM/KOMP-CSD collection are correct, designed to detect changes in a variety of systems, including some problematic events were detected. We have devel- metabolism, dysmorphology, behaviour, cardiovascular, oped a set of quality control (QC) criteria and assays to immunity, visual and auditory response, viability, and screen out affected strains as early as possible following homozygous lethality (Ayadi et al. 2012). Strains are avail- germline transmission of the incorrect alleles. able to the scientific community directly from Sanger Insti- tute while colonies are actively breeding, and from the European Mutant Mouse Archive (Wilkinson et al. 2010)or KOMP Repository (Lloyd 2011) once archived. The primary Materials and Methods phenotypic data are also readily available at the Sanger Mouse Portal (http://www.sanger.ac.uk/mouseportal). The care and use of all mice in this study were in accor- At the time of writing, the EUCOMM/KOMP-CSD ES dance with the UK Home Office regulations, UK Animals clone collection consisted of targeted clones for 12,350 (Scientific Procedures) Act of 1986, and were approved by Fig. 1 EUCOMM/KOMP-CSD allele structure and conversion. The the ‘‘knockout-first’’ allele to a conditional allele (tm1c) and restores EUCOMM/KOMP-CSD allele ‘‘knockout-first’’ allele (tm1a) con- the gene’s activity. Subsequent exposure to Cre recombinase will then tains an IRES:lacZ trapping cassette and a floxed promoter-driven neo delete the floxed exon of the tm1c allele resulting in a frameshift and cassette inserted into the intron of the targeted gene. The presence of null mutation (tm1d). Cre recombinase can also be used to convert the an Engrailed (En2) splice acceptor disrupts gene function, resulting in tm1a allele to the tm1b form and generate a nonconditional lacZ- a lacZ fusion for studying gene expression localisation. Exposure to a tagged null allele without the promoter-driven neo cassette source of Flp recombinase removes the gene trap cassette, converts 123 E. Ryder et al.: EUCOMM/KOMP-CSD Mouse QC the Wellcome Trust Sanger Institute Ethical Review or, where no product was detected, a PCR using two gene- Committee. specific PCRs followed by sequencing. Further details of the tests used at the Sanger Institute, A Minimum Standard for Mouse QC including primer sequences and reaction conditions, can be found in Supplementary Information S1 and also in the The high-throughput nature of the Sanger MGP makes it IKMC knowledge base (http://www.knockoutmouse.org/ impractical to apply a QC strategy based on Southern blot kb/2). analysis. Thus, our QC strategy is centred on a set of PCR-based methods configured to detect abnormalities in the identity of the targeted gene, the presence/absence of Results the 30 loxP site, and the number of vector insertions. Our strategy complies with the IKMC guidelines to include at During the period between September 2006 and November least one test in each of the four different categories 2011, a total of 731 EUCOMM/KOMP ESC clones were (Table 1). microinjected (582 MGP, 94 EUMODIC, and 48 KOMP2- funded) and subsequently achieved germline transmission, High-Throughput Genotyping and QC Tests used of which 632 mouse colonies (86 %) passed QC. by the Sanger Institute MGP Details of the assays that have been performed on the released lines are shown in Fig. 3; not all assays were For rapid and universal detection of potential germline completed on all lines released to the community as the QC transmission of the mutant allele from the initial breeding methods evolved as the MGP has progressed and gained of chimeras (crossed to C57BL/6N Taconic), G1 carriers experience with the KOMP and EUCOMM ES cell are identified by a universal qPCR assay designed to the resource.