Use of Metaphase-Chromosome Transfer for Mammalian Gene Mapping

  • David E. Housman
  • David L. Nelson


The mapping of mammalian chromosomes can be approached by two basic strategies: genetic linkage and physical mapping. The precision with which genetic mapping can be applied to a given chromosomal region is directly related to the number of polymorphic markers available in that region and the spacing of those markers within the region. Physical mapping techniques are limited by the sizes of DNA fragments that can be manipulated, and until recently, these limits were in the range of tens of kilobase pairs of DNA. Recent technical innovations promise the potential for manipulating mammalian DNA segments hundreds of kilobase pairs in length. It is clear that the power of both genetic-linkage and physical-isolation techniques would be enhanced by the ability to isolate and characterize chromosomal segments 105 base pairs or greater in length by a direct genetic technique. Previously, we have suggested a strategy based on metaphase-chromosome transfer to address this issue (Housman et al., 1983). For the past several years, our research efforts have been devoted to development of this strategy. The results presented herein bear on the utility of chromosome transfer both for the physical mapping of chromosomes and for the development of DNA-based markers at intervals of a centimorgan or less within these chromosomal segments.


Selectable Marker Thymidine Kinase Integration Site Recipient Cell Chromosome Transfer 
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  1. Burch, J. W., and McBride, O. W., 1975, Human gene expression in rodent cells after uptake of isolated metaphase chromosomes, Proc. Natl. Acad. Sci. U.S.A. 72: 1797–1801.PubMedCrossRefGoogle Scholar
  2. Capecchi, M. R., 1980, High efficiency transformation by direct microinjection of DNA into cultured mammalian cells, Cell 22: 479–488.PubMedCrossRefGoogle Scholar
  3. Cepko, C. L., Roberts, B. E., and Mulligan, R. C., 1984, Construction and applications of a highly transmissible murine retrovirus shuttle vector, Cell 37: 1053–1062.PubMedCrossRefGoogle Scholar
  4. Chao, M. V., Mellon, P., Charnay, P., Maniatis, T., and Axel, R., 1983, The regulated expression of beta globin genes introduced into mouse erythroleukemia cells, Cell 32: 483–493.PubMedCrossRefGoogle Scholar
  5. Cirullo, R. E., Dana, S., and Wasmuth, J. J., 1983, Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: Interspecific transfer of the human genes encoding leucyl-and asparaginyl-tRNA synthetases, Mol. Cell. Biol. 3: 892–902.PubMedGoogle Scholar
  6. Cone, R. D., and Mulligan, R. C., 1984, High efficiency gene transfer into mammalian cells: Generation of helper-free recombinant retrovirus with broad mammalian host range, Proc. Natl. Acad. Sci. U.S.A. 81: 6349–6353.PubMedCrossRefGoogle Scholar
  7. Degnen, G. E., Miller, I. L., Eisenstadt, J. M., and Adelberg, E. A., 1976, Chromosome-mediated gene transfer between closely related strains of cultured mouse cells, Proc. Natl. Acad. Sci. U.S.A. 73: 2838–2842.PubMedCrossRefGoogle Scholar
  8. Fournier, R. E. K., 1983, Microcell-mediated chromosome transfer, in: Techniques in Somatic Cell Genetics ( J. M. Shay, ed.), pp. 309–327, Plenum Press, New York.Google Scholar
  9. Gerhard, D. S., Kawasaki, E. S., Bancroft, F. C., and Szabo, P., 1981, Localization of a unique gene by direct hybridization in situ, Proc. Natl. Acad. Sci. U.S.A. 78: 3755–3759.PubMedCrossRefGoogle Scholar
  10. Glaser, T., and Housman, D., 1984, Insertion of a selectable marker into various sites on human chromosome #11, 1CSU Short Reports, Vol. 1, pp. 174–175Google Scholar
  11. Miami Winter Symposium. Graham, F. L., and van der Eb, A., 1973, A new technique for the assay of infectivity of human adenovirus 5 DNA, Virology 52: 456–467.Google Scholar
  12. Gusella, J. F., Keys, C., Varsanyi-Breiner, A., Kao, F.-T., Jones, C., Puck, T. T., and Housman, D., 1980, Isolation and localization of DNA segments from specific human chromosomes, Proc. Natl. Acad. Sci. U.S.A. 77: 2829–2833.PubMedCrossRefGoogle Scholar
  13. Gusella, J. F., Jones, C., Kao, F.-T., Housman, D., and Puck, T. T., 1982, Genetic fine-structure mapping in human chromosome 11 by use of repetitive DNA sequences, Proc. Natl. Acad. Sci. U.S.A. 79: 7804–7808.PubMedCrossRefGoogle Scholar
  14. Gusella, J. F., Wexler, N. S., Conneally, P. M., Naylor, S. L., Anderson, M. A., Tanzi, R. E., Watkins, P. C., Ottina, K., Wallace, M. R., Sakaguchi, A. Y., Young, A. B., Shoulson, I., Bonilla, E., and Martin, J. B., 1983, A polymorphic DNA marker genetically linked to Huntington’s disease, Nature (London) 306: 234–238.CrossRefGoogle Scholar
  15. Gusella, J. F., Tanzi, R. E., Anderson, M. A., Hobbs, W., Gibbons, K., Raschtchian, R., Gilliam, T. C., Wallace, M. R., Wexler, N. S., and Conneally, P. M., 1984, DNA markers for nervous system diseases, Science 225: 1320–1326.PubMedCrossRefGoogle Scholar
  16. Haber, D. A., and Schimke, R. T., 1982, Chromosome-mediated transfer and amplification of an altered mouse dihydrofolate reductase gene, Somat. Cell Genet. 8: 499–508.PubMedCrossRefGoogle Scholar
  17. Hartley, J. W., and Rowe, W. P., 1976, Naturally occurring mutine leukemia viruses in wild mice: Characterization of a new “amphotropic” class, J. Virol. 19: 19–25.PubMedGoogle Scholar
  18. Hood, L., Steinmetz, M., and Malissen, D., 1983, Genes of the major histocompatibility complex of the mouse, Annu. Rev. Immunol. 1: 529–568.PubMedCrossRefGoogle Scholar
  19. Housman, D., Nelson, D. L., Albritton, L. M., Minden, M., Wieder, S., and Mulligan, R. C., 1983, Gene mapping by metaphase chromosome transfer: Use of an inserted bacterial gene as a selectable marker, in: Banbury Report No. 14 ( C. T. Caskey and R. L. White, eds.), pp. 197–203, Cold Spring Harbor Press, Cold Spring Harbor, New York.Google Scholar
  20. Hudson, L. D., Erbe, R. W., and Jacoby, L. B., 1980, Expression of the human argininosuccinate synthetase gene in hamster transferents, Proc. Natl. Acad. Sci. U.S.A. 77: 4234–4238.PubMedCrossRefGoogle Scholar
  21. Ishiura, M., Hirose, S., Uchida, T., Hamada, Y., Suzuki, Y., and Okada, Y., 1982, Phage particle-mediated gene transfer to cultured mammalian cells, Mol. Cell. Biol. 2: 607–616.PubMedGoogle Scholar
  22. Klobutcher, L. A., and Ruddle, F. H., 1979, Phenotype stabilisation and integration of transferred material in chromosome-mediated gene transfer, Nature (London) 280: 657–660.CrossRefGoogle Scholar
  23. Klobutcher, L. A., Miller, C. L., and Ruddle, F. H., 1980, Chromosome-mediated gene transfer results in two classes of unstable transformants, Proc. Natl. Acad. Sci. U.S.A. 77: 3610–3614.PubMedCrossRefGoogle Scholar
  24. Law, M. L., Davidson, J. N., and Kao, F.-T., 1982, Isolation of a human repetitive sequence and its application to regional chromosome mapping, Proc. Natl. Acad. Sci. U.S.A. 79: 7390–7394.PubMedCrossRefGoogle Scholar
  25. Lewis, W. H., Srinivasan, P. R., Stokoe, N., and Siminovitch, L., 1980, Parameters governing the transfer of the genes for thymidine kinase and dihydrofolate reductase into mouse cells using metaphase chromosomes or DNA, Somat. Cell Genet. 6: 333–347.PubMedCrossRefGoogle Scholar
  26. Lugo, T. G., and Baker, R. M., 1983, Chromosome-mediated gene transfer of HPRT and APRT in an intraspecific human cell system, Somat. Cell Genet. 9: 175–188.PubMedCrossRefGoogle Scholar
  27. Maitland, N. J., and McDougall, J. K., 1977, Biochemical transformation of mouse cells by fragments of herpes simplex virus DNA, Cell 11: 233–241.PubMedCrossRefGoogle Scholar
  28. Mann, R., Mulligan, R. C., and Baltimore, D., 1983, Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus, Cell 33: 153–159.PubMedCrossRefGoogle Scholar
  29. McBride, O. W., and Ozer, H. L., 1973, Transfer of genetic information by purified metaphase chromosomes, Proc. Natl. Acad. Sci. U.S.A. 70: 1258–1262.PubMedCrossRefGoogle Scholar
  30. Miller, C. L., and Ruddle, F. H., 1978, Co-transfer of human X-linked markers into murine somatic cells via isolated metaphase chromosomes, Proc. Natl. Acad. Sci. U.S.A. 75: 3987–3990.Google Scholar
  31. Mulligan, R. C., 1983, Construction of highly transmissible mammalian cloning vehicles derived from murine retroviruses, in: Experimental Manipulation of Gene Expression ( M. Inouye, ed.), pp. 155–173, Academic Press, New York.Google Scholar
  32. Mulligan, R. C., and Berg, P., 1980, Expression of a bacterial gene in mammalian cells, Science 209: 1422–1427.PubMedCrossRefGoogle Scholar
  33. Murphy, P. D., Miller, C. L., and Ruddle, F. H., 1985, Quantitation of the transgenome size in chromosome-mediated gene transfer lines, Cytogenet. Cell Genet. 39: 125–133.PubMedCrossRefGoogle Scholar
  34. Murray, J. M., Davies, K. E., Harper, P. S., Meredith, L., Mueller, C. R., and Williamson, R., 1982, Linkage relationship of a cloned DNA sequence on the short arm of the X chromosome to Duchenne muscular dystrophy, Nature (London) 300: 69–71.CrossRefGoogle Scholar
  35. Nelson, D. L., Weis, J. H., Przyborski, M. J., Mulligan, R. C., Seidman, J. G., and Housman, D. E., 1984, Metaphase chromosome transfer of introduced selectable markers, J. Mol. Appl. Genet. 2: 563–577.Google Scholar
  36. Nelson, D. L., Przyborski, M. J., Housman, D. E., Seidman, J. G., and Weis, J. H., 1986, Insertion of a retroviral vector between the K and 1 region of the murine histocompatibility complex (submitted).Google Scholar
  37. Oie, H. K., Russell, E. K., Dotson, J. H., Rhoads, J. M., and Gazdar, A. F., 1976, Host-range properties of murine xenotropic and ecotropic type-C viruses, J. Natl. Cancer Inst. 56: 423–426.PubMedGoogle Scholar
  38. Olsen, A. S., McBride, O. W., and Moore, D. E., 1981, Number and size of human X chromosome fragments transferred to mouse cells by chromosome-mediated gene transfer, Mol. Cell. Biol. 1: 439–448.PubMedGoogle Scholar
  39. Pellicer, A., Wigler, M., Axel, R., and Silverstein, S., 1978, The transfer and stable integration of the HSV-thymidine kinase gene into mouse cells, Cell 14: 133–141.PubMedCrossRefGoogle Scholar
  40. Rein, A., Schultz, A. M., Bader, J. P., and Bassin, R. H., 1982, Inhibitors of glycosylation reverse retroviral interference, Virology 119: 185–192.PubMedCrossRefGoogle Scholar
  41. Scangos, G. A., Huttner, K. M., Silverstein, S., and Ruddle, F. H., 1979, Molecular analysis of chromosome-mediated gene transfer, Proc. Natl. Acad. Sci. U.S.A. 76: 3987–3990.PubMedCrossRefGoogle Scholar
  42. Schaffner, W., 1980, Direct transfer of cloned genes from bacteria to mammalian cells, Proc. Natl. Acad. Sci. U.S.A. 77: 2163–2167.PubMedCrossRefGoogle Scholar
  43. Sege, R. D., Kozarsky, K., Nelson, D. L., and Krieger, M., 1984, Expression and regulation of human low-density lipoprotein receptors in Chinese hamster ovary cells, Nature (London) 307: 742–745.CrossRefGoogle Scholar
  44. Shih, C., and Weinberg, R. A., 1982, Isolation of a transforming sequence from a human bladder carcinoma cell line, Cell 29: 161–169.PubMedCrossRefGoogle Scholar
  45. Shih, C., Shilo, B.-Z., Goldfarb, M. P., Dannenberg, A., and Weinberg, R. A., 1979, Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin, Proc. Natl. Acad. Sci. U.S.A. 76: 5714–5718.PubMedCrossRefGoogle Scholar
  46. Shimotohno, K., and Temin, H. M., 1980, No apparent nucleotide sequence specificity in cellular DNA juxtaposed to retrovirus proviruses, Proc. Natl. Acad. Sci. U.S.A. 77: 7357–7361.PubMedCrossRefGoogle Scholar
  47. Shimotohno, K., and Temin, H. M., 1981, Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus, Cell 26: 67–77.PubMedCrossRefGoogle Scholar
  48. Shoemaker, C. S., Goff, S., Gilboa, E., Paskind, M., Mitra, S. W., and Baltimore, D., 1980, Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: Implication for retrovirus integration, Proc. Natl. Acad. Sci. U.S.A. 77: 3932–3936.PubMedCrossRefGoogle Scholar
  49. Shoemaker, C., Hoffmann, J., Goff, S. P., and Baltimore, D., 1981, Intramolecular integration within Moloney murine leukemia virus DNA, J. Virol. 40: 164–172.PubMedGoogle Scholar
  50. Shows, T. B., Naylor, S. L., and Sakaguchi, A. Y., 1982, Mapping the human genome, cloned genes, DNA polymorphisms, and inherited disease, in: Advances in Human Genetics, Vol. 12 ( H. Harris and K. Hirschhorn, eds.), pp. 341–452, Plenum Press, New York.Google Scholar
  51. Simmons, T., Lipman, M., and Hodge, L. D., 1978, Uptake and early fate of metaphase chro- mosomes ingested by the Wi-L2 human lymphoid cell line, Somat. Cell Genet. 4: 55–76.PubMedCrossRefGoogle Scholar
  52. Southern, E. M., 1975, Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol. 98: 503–517.PubMedCrossRefGoogle Scholar
  53. Southern, P. J., and Berg, P., 1982, Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter, J. Mol. Appl. Genet. 1: 327–341.PubMedGoogle Scholar
  54. Szybalski, W., Szybalska, E. H., and Ragni, G., 1962, Genetic studies with human cell lines, Natl. Cancer Inst. Monogr. 7: 75–89.Google Scholar
  55. Tabin, C. J., Hoffman, J. W., Goff, S. P., and Weinberg, R. A., 1982, Adaptation of a retrovirus as a eukaryotic vector transmitting the herpes simplex virus thymidine kinase gene, Mol. Cell. Biol. 2: 426–436.PubMedGoogle Scholar
  56. Temin, H., and Baltimore, D., 1972, RNA-directed DNA synthesis and RNA tumor viruses, Adv. Virus Res. 17.Google Scholar
  57. Weis, J. H., Nelson, D. L., Przyborski, M. J., Mulligan, R. C., Chaplin, D. D., Housman, D. E., and Seidman, J. G., 1984, Eukaryotic chromosome transfer: Linkage of the murine major histocompatibility complex to an inserted dominant selectable marker, Proc. Natl. Acad. Sci. U.S.A. 81: 4879–4883.PubMedCrossRefGoogle Scholar
  58. Weis, J. H., Seidman, J. G., Housman, D. E., and Nelson, D. L., 1986, Eukaryotic chromosome transfer: Production of a murine specific cosmid library from a Neo’ linked fragment of murine chromosome 17, Mol. Cell. Biol. 6: 441–451.PubMedGoogle Scholar
  59. Wigler, M., Silverman, S. Lee, L.-S., Pellicer, A., Cheng, Y., and Axel, R., 1977, Transfer of purified herpes virus thymidine kinase genes to cultured mouse cells, Cell 14: 725–731.Google Scholar
  60. Wigler, M., Pellicer, S., Silverstein, S., and Axel, R., 1978, Biochemical transfer of single-copy eukaryotic genes using total cellular DNA as donor, Cell 14: 725–731.PubMedCrossRefGoogle Scholar
  61. Wigler, M., Pellicer, S., Silverstein, S., Axel, R., Urlaub, G., and Chasin, L., 1979a, DNA-mediated transfer of the adenine phosphoribosyl transferase locus into mammalian cells, Proc. Natl. Acad. Sci. U.S.A. 76: 1373–1376.PubMedCrossRefGoogle Scholar
  62. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y., and Axel, R., 1979b, Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells, Cell 14: 725–731.CrossRefGoogle Scholar
  63. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R., 1979c, Transformation of mammalian cells with genes from prokaryotes and eukaryotes, Cell 16: 777–785.PubMedCrossRefGoogle Scholar
  64. Wigler, M., Perucho, M., Kurtz, K.. Dana, S., Pellicer, A., Axel, R., and Silverstein, S., 1980, Transformation of mammalian cells with an amplifiable dominant-acting gene, Proc. Natl. Acad. Sci. U.S.A. 77: 3567–3570.Google Scholar
  65. Willecke, K., and Ruddle, F. H., 1975, Transfer of the human gene for hypoxanthine phosphoribosyltransferase via isolated human metaphase chromosomes into mouse L-cells, Proc. Natl. Acad. Sci. U.S.A. 72: 1792–1796.PubMedCrossRefGoogle Scholar
  66. Willecke, K., Lange, R., Kruger, A., and Reber, T., 1976, Cotransfer of two linked human genes into cultured mouse cells, Proc. Natl. Acad. Sci. U.S.A. 73: 1274–1278.PubMedCrossRefGoogle Scholar
  67. Willing, M. C., Nienhuis, A. W., and Anderson, W. F., 1976, Selective activation of human beta but not gamma globin gene in human fibroblast x mouse erythroleukemia cell hybrids, Nature (London) 277: 534–538.CrossRefGoogle Scholar
  68. Wright, S., deBoer, E., Grosveld, F. G., and Flavell, R. A., 1983, Regulated expression of the human beta globin gene family in murine erythroleukaemia cells, Nature (London) 305: 333–336.CrossRefGoogle Scholar
  69. Wullems, G. J., van der Horst, J., and Bootsma, D., 1976, Transfer of the human X chromosome to human—Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes, Somat. Cell Genet. 2: 359–371.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • David E. Housman
    • 1
  • David L. Nelson
    • 2
  1. 1.Center for Cancer Research, Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Laboratory of Molecular Genetics, National Institute of Neurological and Communicative Disorders and StrokeNational Institutes of HealthBethesdaUSA

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