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Functional genomics in mice by tagged sequence mutagenesis

Nature Genetics volume 16, pages 338–344 (1997)Cite this article

Abstract

Most mammalian genes will soon be characterized as cDNA sequences with little information about their function. To utilize this sequence information for large-scale functional studies, a gene trap retrovirus shuttle vector has been developed to disrupt genes expressed in murine embryonic stem (ES) cells. A library of mutant clones was isolated, and regions of genomic DMA adjacent to 400 independent provirus inserts were cloned and sequenced. The flanking sequences, designated ‘promoter-proximal sequence tags’, or PSTs, identified 63 specific genes and anonymous cDNAs disrupted as a result of virus integration. The efficiency of tagged sequence mutagenesis suggests that many of the 10,000–20,000 genes expressed in ES cells can be targeted, providing defined mutations for the analysis of gene functions in vivo. In addition, PSTs provide the first expressed sequence tags derived from genomic DNA, and define gene features such as exon boundaries and promoters that are missing from cDNA sequences.

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References

  1. Gibbs, R.A. Pressing ahead with human genome sequencing. Nature Genet. 11, 121–125 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Oliver, S.G. From DNA sequence to biological function. Nature 379, 597–600 (1996).

    Article  CAS  PubMed  Google Scholar 

  3. McKusick, V.A., Inheritance in Man: Catalogue of Autosoma Dominant, Autosomal Recessive, and X-Linked Phenotypes 1626 (John Hopkins University, Baltimore, 1988).

  4. Green, M.C. Catalog of mutant genes and polymorphic loci, in Genetic Variants and Strains of the Laboratory Mouse (eds Lyon, M. F. & Searle, A.G.) 12–403. (Oxford University, Oxford, England, 1989).

  5. Reith, A.D. & Berstein, A. Molecular basis of mouse developmental mutants. Genes Devel. 5, 1115–1123 (1991).

    Article  CAS  PubMed  Google Scholar 

  6. Capecchi, M.R. Altering the genome by homologous recombination. Science 244, 1288–1292 (1989).

    Article  CAS  PubMed  Google Scholar 

  7. Brandon, E.P., Idzerda, R.L. & S., M.G. Targeting the mouse genome: a compendium of knockouts (part I). Curr. Blol. 5, 625–634 (1995).

    Article  CAS  Google Scholar 

  8. Gossler, A., Joyner, A.L., Rossant, J. & Skarnes, W.C. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244, 463–465 (1989).

    Article  CAS  PubMed  Google Scholar 

  9. 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  PubMed  Google Scholar 

  10. von Melchner, H. et al. Selective disruption of genes expressed in totipotent embryonal stem cells. Genes Dev. 6, 919–927 (1992).

    Article  CAS  PubMed  Google Scholar 

  11. Skarnes, W.C., Auerbach, B.A. & Joyner, A. A gene trap approach in mouse embryonic stem cells: the lacZ reporter is activated by splicing reflects endogenous gene expression, and is mutagenic in mice.Genes Dev. 6, 903–918 (1992).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Scherer, C.A., Chen, J., Nachabeh, A., Hopkins, N. & Ruley, H.E. Transcriptional specif icity of the pluripotent embryonic stem cell. Cell Growth Differ. 7, 1393–1401 (1996).

    CAS  PubMed  Google Scholar 

  14. Forrester, L.M. et al. An induction gene trap screen in embryonic stem cells: identification of genes that respond to retinoic acid in vitro. Proc. Natl. Acad. Sci. USA 93, 1677–1682 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Reddy, S., Rayburn, H., von Melchner, H. & Ruley, H.E. Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes. Proc. Natl. Acad. Sci. USA 89, 6721–6725 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wurst, W. et al. A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice. Genetics 139, 889–899 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Nussbaum, R.L., Lesko, J.G., Lewis, R.A., Ledbetter, S.A. & Ledbetter, D.H. Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation. Proc. Natl. Acad. Sci. USA 84, 6521–6525 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  20. 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  PubMed  Google Scholar 

  21. Adams, M.D. et al. Sequence identification of 2,375 human brain genes. Nature 13, 632–634 (1992).

    Article  Google Scholar 

  22. Adams, M.D. et al. Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1643–1651 (1991).

    Article  Google Scholar 

  23. Adams, M.D., Kerlavage, A.R., Fields, C. & Venter, J.C. 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet. 4, 256–267 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Okubo, K. et al. Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nature Genet. 2, 173–179 (1992).

    Article  CAS  PubMed  Google Scholar 

  25. Nehls, M., Pfeifer, D., Micklem, G., Schmoor, C. & Boehm, T. The sequence complexity of exons trapped from the mouse genome. Curr. Biol. 4, 983–989 (1994).

    Article  CAS  PubMed  Google Scholar 

  26. Crozat, A., Aman, P., Mandahl, N. & Ron, D. Fusion of CHOP to a novel RNA–binding protein in human myxoid liposarcoma. Nature 363, 640–644 (1993).

    Article  CAS  PubMed  Google Scholar 

  27. Rabbitts, T.H., Forster, A., Larson, R. & Nathan, P. Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nature Genet. 4, 175–180 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. Zucman, J. et al. Combinatorial generation of variable fusion proteins in the Ewing family of tumours. EMBO J. 12, 4481–4487 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zucman, J. et al. EWS and ATF-1 gene fusion induced by t(12;22) translocation in malignant melanoma of soft parts. Nature Genet. 4, 341–345 (1993).

    Article  CAS  PubMed  Google Scholar 

  30. Llamazares, S. et al, polo encodes a protein kinase homolog required for mitosis in Drosophila . Genes Dev. 5, 2153–2165 (1991).

    Article  CAS  PubMed  Google Scholar 

  31. Clay, F.J., McEwen, S., Bertoncello, I., Wilks, A.F. & Dunn, A.R. Identification and cloning of a protein kinase encoding mouse gene Plk, related to the polo gene of Drosophila . Proc. Natl. Acad. Sci. USA 90, 4882–1886 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fenton, B. & Glover, D.M. A conserved mitotic kinase active at late anaphase-telophase in syncytial Drosophila embryos. Nature 363, 637–640 (1993).

    Article  CAS  PubMed  Google Scholar 

  33. Lake, R.J. & Jelinek, W.R. Cell cycle- and terminal differentiation-associated regulation of the mouse mRNA encoding a conserved mitotic protein kinase. Mol. Cell. Biol. 13, 7793–7801 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Rendahl, K.G., Jones, K.R., Kulkarni, S.J., Bagully, S.H. & Hall, J.C. The dissonance mutation at the no-on-transient–A locus of D. melanogaster: genetic control of courtship song and visual behaviors by a protein with putative RNA-binding motifs J. Neurosci. 12, 390–407 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yang, Y.S. et al. NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss. Mol. Cell. Biol. 13, 5593–5603 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Duncan, R. et al. A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev. 8, 465–480 (1994).

    Article  CAS  PubMed  Google Scholar 

  37. Coccia, E.M. et al. Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development. Mol. Cell. Biol. 12, 3514–3521 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. DeGregori, J. et al. A murine homolog of the yeast RNA1 gene is required for postimplantation development. Genes Dev. 8, 265–276 (1993).

    Article  Google Scholar 

  39. Chen, J. et al. Germline inactivation of the murine eck receptor tyrosine kinase by gene trap retroviral insertion. Oncogene 12, 979–988 (1996).

    CAS  PubMed  Google Scholar 

  40. Lewin, B. Units of transcription and translation: sequence components of heterogeneous nuclear RNA and messenger RNA. Cell 4, 77–93 (1975).

    Article  CAS  PubMed  Google Scholar 

  41. 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  PubMed  Google Scholar 

  42. Deng, J.M. & Behringer, R.R. An insertional mutation in the BTF3 transcription factor gene leads to an early postimplantation lethality in mice. Transgenic Res. 4, 264–269 (1995).

    Article  CAS  PubMed  Google Scholar 

  43. Gasca, S., Hill, D.P., Klingensmith, J. & Rossant, J. Characterization of a gene trap insertion into a novel gene, cordon-bleu, expressed in axial structures of the gastrulating mouse embryo. Dev. Genet. 17, 141–154 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Takeuchi, T. et al. Gene trap capture of a novel mouse gene, jumonji, required for neural tube formation. Genes Dev. 9, 1211–1222 (1995).

    Article  CAS  PubMed  Google Scholar 

  45. Hutchison, C.A. III, Hardies, S.C., Loeb, D.D., Shehee, W.R & Edgell, M.H. Lines and related retroposons: long interspersed repeated sequences in the eucaryotic genome. in Mobile DNA (eds Berg, D.E. & Howe, M.M.) 593–617 (American Society of Microbiology, Washington, D.C., 1989).

    Google Scholar 

  46. Shih, C.C., Stoye, J.P. & Coffin, J.M. Highly preferred targets for retrovirus integration. Cell 53, 531–537 (1988).

    Article  CAS  PubMed  Google Scholar 

  47. Sandmeyer, S.B., Hansen, L.J. & Chalker, D.L. Integration specificity of retrotransposons and retroviruses. Annu. Rev. Genet. 24, 491–518 (1990).

    Article  CAS  PubMed  Google Scholar 

  48. Monk, M., Boubelik, M. & Lehnert, S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371–382 (1987).

    CAS  PubMed  Google Scholar 

  49. Kafri, T. et al. Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev. 6, 705–714 (1992).

    Article  CAS  PubMed  Google Scholar 

  50. Lowe, S.W., Ruley, H.E., Jacks, T. & Housman, D.E. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74, 957–967 (1993).

    Article  CAS  PubMed  Google Scholar 

  51. Lowe, S.W., Schmitt, E.M., Smith, S.W., Osborne, B.A. & Jacks, T. T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362, 847–849 (1993).

    Article  CAS  PubMed  Google Scholar 

  52. Kastan, M.B. et al. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587–597 (1992).

    Article  CAS  PubMed  Google Scholar 

  53. Mortensen, R.M., Conner, D.A., Chao, S., Geisterfer-Lowrance, A.A. & Seidman, J.G. Production of homozygous mutant ES cells with a single targeting construct. Mol. Cell. Biol. 12, 2391–2395 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Yotov, W.V. & St-Arnaud, R. Differential splicing-in of a proline-rich exon converts αNACintoa muscle-specific transcription factor. Genes Dev. 10, 1763–1772 (1996).

    Article  CAS  PubMed  Google Scholar 

  55. Ramirez-Solis, R., Liu, P. & Bradley, A. Chromosome engineering in mice. Nature 378, 720–724 (1995).

    Article  CAS  PubMed  Google Scholar 

  56. Chen, J. et al. Retrovirus gene traps. Meth Mol Genet 4, 123–140 (1994).

    CAS  Google Scholar 

  57. Hicks, G.G. et al. Retrovirus gene traps. Methods Enzymol 254, 263–275 (1995).

    Article  CAS  PubMed  Google Scholar 

  58. Hsiao, K. A fast and simple procedure for sequencing double stranded DNA with Sequenase.Nucleic Acids Res. 19, 2787 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Vanderbilt University School of Medicine, 116121st Avenue South, Nashville, Tennessee, 37232-2363, USA

    Geoffrey G. Hicks, Er-gang Shi, Xuan-Mei Li, Chun-Hua Li, Maciej Pawlak & H. Earl Ruley

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Hicks, G., Shi, Eg., Li, XM. et al. Functional genomics in mice by tagged sequence mutagenesis. Nat Genet 16, 338–344 (1997). https://doi.org/10.1038/ng0897-338

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