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Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells

Nature volume 392, pages 608–611 (1998)Cite this article

Abstract

The dramatic increase in sequence information in the form of expressed sequence tags (ESTs)1 and genomic sequence has created a ‘gene function gap’, with the identification of new genes faroutpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms2,3. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.

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Figure 1: Vector design for 3′ trapping.
Figure 2: Targeting of VICTR20 to the second intron of the Hprt locus.
Figure 3: VICTR20 has integrated into the first intron of the btk gene.
Figure 4: Expression of ‘unknowns’.

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References

  1. Adams, M. D.et al. Complementary DNA sequencing: “Expressed sequence tags” and the Human Genome Project. Science 252, 1651–1656 (1991).

    Article  ADS  CAS  Google Scholar 

  2. Plasterk, R. H. Reverse genetics of Caenorhabditis elegans. Bioessays 14, 629–633 (1992).

    Article  CAS  Google Scholar 

  3. Spradling, A. C.et al. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc. Natl Acad. Sci. USA 92, 10824–10830 (1995).

    Article  ADS  CAS  Google Scholar 

  4. Gossler, A. & Zachgo, J. in Gene Targeting: A Practical Approach(ed. Joyner, A. L.) 181–227 (Oxford Univ. Press, New York, (1993).

    Google Scholar 

  5. Friedrich, G. & Soriano, P. Insertional mutagenesis by retroviruses and promoter traps in embryonic stem cells. Meth. Enzymol. 225, 681–701 (1993).

    Article  CAS  Google Scholar 

  6. Chen, Z., Friedrich, G. A. & Soriano, P. Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice. Genes Dev. 8, 2293–2301 (1994).

    Article  CAS  Google Scholar 

  7. Zambrowicz, B. P.et al. Disruption of overlapping transcripts in the ROSA βgeo 26 gene trap strain leads to widespread expression of βgalactosidase in mouse embryos and hematopoietic cells. Proc. Natl Acad. Sci. USA 94, 3789–3794 (1997).

    Article  ADS  CAS  Google Scholar 

  8. Townley, D. J., Avery, B. J., Rosen, B. & Skarnes, W. C. Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice. Genome Res. 7, 293–298 (1997).

    Article  CAS  Google Scholar 

  9. Hicks, G. G.et al. Functional genomics in mice by tagged sequence mutagenesis. Nature Genet. 16, 338–344 (1997).

    Article  CAS  Google Scholar 

  10. Friedrich, G. & Soriano, P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513–1523 (1991).

    Article  CAS  Google Scholar 

  11. Skarnes, W., Moss, J., Hurtley, S. & Beddington, R. Capturing genes encoding membrane and secreted proteins important for mouse development. Proc. Natl Acad. Sci. USA 92, 6592–6596 (1995).

    Article  ADS  CAS  Google Scholar 

  12. Altschul, S. F., Gish, W., Miller, W., Meyers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  Google Scholar 

  13. Oeltjen, J. C.et al. Large-scale comparative sequence analysis of the human murine Bruton's tyrosine kinase loci reveals conserved regulatory domains. Genome Res. 7, 315–329 (1997).

    Article  CAS  Google Scholar 

  14. de Weers, M.et al. The Bruton's tyrosine kinase gene is expressed throughout B cell differentiation, from early precursor B cell stages preceding immunoglobulin gene rearrangement up to mature B cell stages. Eur. J. Immunol. 23, 3109–3114 (1993).

    Article  CAS  Google Scholar 

  15. Chang, W., Hubbard, C., Friedel, C. & Ruley, E. Enrichment of insertional mutants following retrovirus gene trap selection. Virology 193, 737–747 (1993).

    Article  CAS  Google Scholar 

  16. Withers-Ward, E. S., Kitamura, Y., Barnes, J. P. & Coffin, J. M. Distribution of targets for avian retrovirus DNA integration in vivo. Genes Dev. 8, 1473–1487 (1994).

    Article  CAS  Google Scholar 

  17. Craigie, R. Hotspots and warm spots: integration specificity of retroelements. Trends Genet. 8, 187–190 (1992).

    Article  CAS  Google Scholar 

  18. Soriano, P., Friedrich, G. & Lawinger, P. Promoter interaction in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J. Virol. 65, 2314–2319 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhu, B., Bogue, M. A., Lim, D.-S., Hasty, P. & Roth, D. B. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86, 379–389 (1996).

    Article  CAS  Google Scholar 

  20. Hasty, P., Rivera-Perez, J., Chang, C. & Bradley, A. Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells. Mol. Cell. Biol. 11, 4509–4517 (1991).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Bradley for ideas; M. Brandt, J. Fitzgibbons, C. Mossel, M. Newhouse, D. Potter and T. Smith for technical assistance; J. D. Wallace and A. Benjamin for tissue culture; D. Becer, L. Grocki, D. Leibham, T. Malley and S. Patel for automation and sequencing; B. Kohlhauf for computer support; and P. Hasty, R. Burgess, A. Turner and M. Nehls for discussion.

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  1. Lexicon Genetics, 4000 Research Forest Drive, The Woodlands, 77381, Texas, USA

    Brian P. Zambrowicz, Glenn A. Friedrich, Eric C. Buxton, Stan L. Lilleberg, Christophe Person & Arthur T. Sands

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  1. Brian P. Zambrowicz
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  2. Glenn A. Friedrich
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Zambrowicz, B., Friedrich, G., Buxton, E. et al. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature 392, 608–611 (1998). https://doi.org/10.1038/33423

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