Archived Page: Mouse developmental genetics and ES cell mutagenesis

From 2003 to 2014 Bill Skarnes led the work of the Mouse developmental genetics and ES cell mutagenesis team. The team changed research focus in 2014 to concentrate on human stem cells and their current work is described on a new research page: Stem Cell Engineering. This page is being maintained to provide an historical record of the team’s mouse research.

The Mouse developmental genetics and ES cells mutagenesis team employed high-throughput mutagenesis strategies for the functional annotation of genes in mouse embryonic stem (ES) cells and in mice.

The use of mouse embryonic stem (ES) cells to elucidate diverse areas in biology offered great potential and was under-utilised. ES cells represented a genetically tractable model system to study basic cell biological and developmental processes on a genome-wide scale. Our work established gene-based phenotype-driven screens in mouse ES cells to elucidate genetic pathways required for ES cell self-renewal and pluripotency. We developed a more complete understanding of the programme of events associated with ES cell differentiation that suggested ways to direct mouse ES cells along specific cell lineages. Our ultimate goal was to develop techniques that could be used engineer and reprogramme human stem cells to provide appropriate cell types for somatic cell replacement therapies for the treatment of human disease. (See the new project: Stem Cell Engineering)

[Genome Research Limited]

Background

The highly conserved human and mouse genomes comprise about 20,000 protein-coding genes of mostly unknown function. As part of an international programme aimed at mutating all protein-coding genes in the mouse, we used a combination of gene trapping and gene targeting technologies to generate a large public resource of reporter-tagged mutations in mouse ES cells for the functional annotation of the mouse genome. Mutant ES cells from this resource support the Sanger Institute Mouse Genetics Project and are available to the scientific community without restriction.

Research

Our aims

Formation of mesoderm in a gastrulating mouse embryo.

Formation of mesoderm in a gastrulating mouse embryo.

The Mouse developmental genetics and ES cell mutagenesis team had two major aims:

  • The first was to produce mutant mouse embryonic stem (ES) cell resources for the scientific community.
  • The second was to use these ES cell resources to understand the underlying genetic basis of early cell fate decisions in the mammalian embryo.

Our approach

A high-throughput gene targeting pipeline was established at the Wellcome Trust Sanger Institute to deliver conditional targeted mutations in more than 10,000 genes in C57BL/6N ES cells. This effort required the construction of vectors and the production of targeted mouse ES cells lines on an unprecedented scale. The Sanger Institute is a member of the International Knockout Mouse Consortium (IKMC) and contributed resources to the EUCOMM and KOMP projects.

The production of large-scale genetic screens in ES cells was hampered by the difficulty in generating homozygous mutant cells. Moreover, genes essential for the growth of stem cells required conditional strategies to recover and study loss-of-function mutations. Taking advantage of high-throughput gene targeting technology, we developed high-efficiency strategies for the generation of conditional homozygous mutant ES cells by serial targeting and the use of ligand-inducible Cre recombinase. This approach permitted the study of genes required for stem cell pluripotency and self-renewal.

Functional analysis chromatin proteins in early mouse development

It is abundantly clear that packaging of genomic DNA is not equivalent in all cell types and various epigenetic modifications profoundly influence the transcriptional competence of genes and developmental potential of cells. We examined the function of chromatin-associated proteins in undifferentiated and differentiated ES cell cultures. These included components of the SWI/SNF chromatin-remodeling complexes, Polycomb group protein complexes and the nucleosome remodelling and deactylation (NuRD) complexes.

Proteomic analysis of chromatin proteins in ES cells

As part of the EUTRACC consortium, we developed and validated a novel strategy for tagging of genes in ES cells for tandem-affinity purification-mass spectrometry (TAP-MS) analysis. This strategy ensured the expression of tagged proteins at endogenous levels and the generation of mice from tagged ES cells that could be used to confirm the normal activity of the tagged protein. Protein-protein and protein-DNA interactions of selected chromatin proteins were analysed also.

Sanger Institute Gene Trap Resource

As a member of the International Gene Trap Consortium (IGTC), we used gene trapping to generate random insertional mutations in mouse ES cells for immediate molecular characterisation. The Sanger Institute Gene Trap Resource (SIGTR) contains more than 10,000 characterised gene trap insertions in 129P2 ES cells. SIGTR gene trap ES cell lines are available upon request from the University of California at Davis.

Resources

Collaborations

  • International Knockout Mouse Consortium (IKMC)
    The International Knockout Mouse Consortium (IKMC) has established a common webportal for the dissemination of mutant ES cell resources to the scientific community.
  • European Conditional Mouse Mutagenesis (EUCOMM)
    Aims to generate a collection of up to 13,000 mutated genes in mouse C57BL/6N embryonic stem (ES) cells using conditional gene trapping and gene targeting approaches.
  • The Knockout Mouse Project (KOMP)
    Is a trans-NIH initiative that aims to generate a comprehensive and public resource comprised of mouse embryonic stem (ES) cells containing a null mutation in every gene in the mouse genome.
  • International Gene Trap Consortium (IGTC)
    A collaboration of laboratories around the world working together to generate a public library of mutated murine ES cell lines, which can be obtained on a non-collaborative basis by scientists interested in generating reporter-tagged, loss-of-function mutations in mice.
  • European Transcriptome, Regulome & Cellular Commitment Consortium (EUTRACC)
    Aims to determine the regulation of the genome by mapping the regulatory pathways and networks of transcription factors that control cellular functions.
  • Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.

    Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X and Skarnes WC

    Nature methods 2014;11;4;399-402

  • The International Mouse Phenotyping Consortium Web Portal, a unified point of access for knockout mice and related phenotyping data.

    Koscielny G, Yaikhom G, Iyer V, Meehan TF, Morgan H, Atienza-Herrero J, Blake A, Chen CK, Easty R, Di Fenza A, Fiegel T, Grifiths M, Horne A, Karp NA, Kurbatova N, Mason JC, Matthews P, Oakley DJ, Qazi A, Regnart J, Retha A, Santos LA, Sneddon DJ, Warren J, Westerberg H, Wilson RJ, Melvin DG, Smedley D, Brown SD, Flicek P, Skarnes WC, Mallon AM and Parkinson H

    Nucleic acids research 2014;42;Database issue;D802-9

  • Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.

    White JK, Gerdin AK, Karp NA, Ryder E, Buljan M, Bussell JN, Salisbury J, Clare S, Ingham NJ, Podrini C, Houghton R, Estabel J, Bottomley JR, Melvin DG, Sunter D, Adams NC, Sanger Institute Mouse Genetics Project, Tannahill D, Logan DW, Macarthur DG, Flint J, Mahajan VB, Tsang SH, Smyth I, Watt FM, Skarnes WC, Dougan G, Adams DJ, Ramirez-Solis R, Bradley A and Steel KP

    Cell 2013;154;2;452-64

  • A conditional knockout resource for the genome-wide study of mouse gene function.

    Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V, Mujica AO, Thomas M, Harrow J, Cox T, Jackson D, Severin J, Biggs P, Fu J, Nefedov M, de Jong PJ, Stewart AF and Bradley A

    Nature 2011;474;7351;337-42

  • Bi-allelic gene targeting in mouse embryonic stem cells.

    Tate PH and Skarnes WC

    Methods (San Diego, Calif.) 2011;53;4;331-8

  • The IKMC web portal: a central point of entry to data and resources from the International Knockout Mouse Consortium.

    Ringwald M, Iyer V, Mason JC, Stone KR, Tadepally HD, Kadin JA, Bult CJ, Eppig JT, Oakley DJ, Briois S, Stupka E, Maselli V, Smedley D, Liu S, Hansen J, Baldock R, Hicks GG and Skarnes WC

    Nucleic acids research 2011;39;Database issue;D849-55

  • Dual RMCE for efficient re-engineering of mouse mutant alleles.

    Osterwalder M, Galli A, Rosen B, Skarnes WC, Zeller R and Lopez-Rios J

    Nature methods 2010;7;11;893-5

  • Floxin, a resource for genetically engineering mouse ESCs.

    Singla V, Hunkapiller J, Santos N, Seol AD, Norman AR, Wakenight P, Skarnes WC and Reiter JF

    Nature methods 2010;7;1;50-2

  • Agouti C57BL/6N embryonic stem cells for mouse genetic resources.

    Pettitt SJ, Liang Q, Rairdan XY, Moran JL, Prosser HM, Beier DR, Lloyd KC, Bradley A and Skarnes WC

    Nature methods 2009;6;7;493-5

  • Extensive genomic copy number variation in embryonic stem cells.

    Liang Q, Conte N, Skarnes WC and Bradley A

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;45;17453-6

  • Gene trapping in mouse embryonic stem cells.

    Brennan J and Skarnes WC

    Methods in molecular biology (Clifton, N.J.) 2008;461;133-48

  • Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells.

    Nord AS, Vranizan K, Tingley W, Zambon AC, Hanspers K, Fong LG, Hu Y, Bacchetti P, Ferrin TE, Babbitt PC, Doniger SW, Skarnes WC, Young SG and Conklin BR

    PloS one 2007;2;7;e617

  • The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse.

    Nord AS, Chang PJ, Conklin BR, Cox AV, Harper CA, Hicks GG, Huang CC, Johns SJ, Kawamoto M, Liu S, Meng EC, Morris JH, Rossant J, Ruiz P, Skarnes WC, Soriano P, Stanford WL, Stryke D, von Melchner H, Wurst W, Yamamura K, Young SG, Babbitt PC and Ferrin TE

    Nucleic acids research 2006;34;Database issue;D642-8

  • Two ways to trap a gene in mice.

    Skarnes WC

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;37;13001-2

  • The knockout mouse project.

    Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS, Dove WF, Duyk G, Dymecki S, Eppig JT, Grieder FB, Heintz N, Hicks G, Insel TR, Joyner A, Koller BH, Lloyd KC, Magnuson T, Moore MW, Nagy A, Pollock JD, Roses AD, Sands AT, Seed B, Skarnes WC, Snoddy J, Soriano P, Stewart DJ, Stewart F, Stillman B, Varmus H, Varticovski L, Verma IM, Vogt TF, von Melchner H, Witkowski J, Woychik RP, Wurst W, Yancopoulos GD, Young SG and Zambrowicz B

    Nature genetics 2004;36;9;921-4

  • A public gene trap resource for mouse functional genomics.

    Skarnes WC, von Melchner H, Wurst W, Hicks G, Nord AS, Cox T, Young SG, Ruiz P, Soriano P, Tessier-Lavigne M, Conklin BR, Stanford WL, Rossant J and International Gene Trap Consortium

    Nature genetics 2004;36;6;543-4

  • BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

    Stryke D, Kawamoto M, Huang CC, Johns SJ, King LA, Harper CA, Meng EC, Lee RE, Yee A, L'Italien L, Chuang PT, Young SG, Skarnes WC, Babbitt PC and Ferrin TE

    Nucleic acids research 2003;31;1;278-81

  • Functional analysis of secreted and transmembrane proteins critical to mouse development.

    Mitchell KJ, Pinson KI, Kelly OG, Brennan J, Zupicich J, Scherz P, Leighton PA, Goodrich LV, Lu X, Avery BJ, Tate P, Dill K, Pangilinan E, Wakenight P, Tessier-Lavigne M and Skarnes WC

    Nature genetics 2001;28;3;241-9

  • Defining brain wiring patterns and mechanisms through gene trapping in mice.

    Leighton PA, Mitchell KJ, Goodrich LV, Lu X, Pinson K, Scherz P, Skarnes WC and Tessier-Lavigne M

    Nature 2001;410;6825;174-9

  • Gene trapping methods for the identification and functional analysis of cell surface proteins in mice.

    Skarnes WC

    Methods in enzymology 2000;328;592-615

  • Capturing novel mouse genes encoding chromosomal and other nuclear proteins.

    Tate P, Lee M, Tweedie S, Skarnes WC and Bickmore WA

    Journal of cell science 1998;111 ( Pt 17);2575-85

  • Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice.

    Townley DJ, Avery BJ, Rosen B and Skarnes WC

    Genome research 1997;7;3;293-8

  • Capturing genes encoding membrane and secreted proteins important for mouse development.

    Skarnes WC, Moss JE, Hurtley SM and Beddington RS

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;14;6592-6

  • The identification of new genes: gene trapping in transgenic mice.

    Skarnes WC

    Current opinion in biotechnology 1993;4;6;684-9

  • A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice.

    Skarnes WC, Auerbach BA and Joyner AL

    Genes & development 1992;6;6;903-18

  • Entrapment vectors: a new tool for mammalian genetics.

    Skarnes WC

    Bio/technology (Nature Publishing Company) 1990;8;9;827-31

  • Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.

    Gossler A, Joyner AL, Rossant J and Skarnes WC

    Science (New York, N.Y.) 1989;244;4903;463-5

Team

No team members listed

* quick link - http://q.sanger.ac.uk/mousedev