Monolayer Enucleation of Colcemid-Treated Human Cells and Polyethylene Glycol 400-Mediated Fusion of Microkaryoplasts (Microcells) with Whole Cells

  • Andrew H. CrenshawJr.
  • Leonard R. Murrell


Modern somatic cell genetics began with the observation that hybridization can be achieved by cell to cell fusion in culture (Barski et al., 1961). In recent years, this new era in the study of gene expression has produced many advances in hybridization technology. Hybridization techniques can be grouped into two general classes: (1) whole genome transfer, involving cell to cell fusion, and (2) partial genome transfer, involving the introduction of genome fragments into whole cells. Mouse-human whole-cell hybridization has been used extensively for human gene mapping (Ruddle and Creagan, 1975; McKusick and Ruddle, 1977) and has relied on the fact that human chromosomes are spontaneously eliminated from these hybrids. Lengthy cultivation, recloning, and analysis are necessary before accurate chromosome assignment is possible (Weiss and Green, 1967).


Mitotic Spindle Condensed Chromosome Cell Fragment Complete Growth Medium Rodent Cell 
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  1. Barski, G., Sorieul, S., and Cornefert, F., 1961, Hybrid type cells in combined cultures of two different mammalian cell strains, J. Natl. Cancer Inst. 26:1269–1291.PubMedGoogle Scholar
  2. Blose, S. H., and Chacko, S., 1976, Rings of intermediate (100 A) filament bundles in the perinuclear region of vascular endothelial cells, J. Cell Biol. 70;459–466.PubMedCrossRefGoogle Scholar
  3. Borisy, G. G., and Taylor, E. W., 1967a, The mechanism of action of colchicine: I. Binding of colchicine-3H to cellular protein, J. Cell Biol. 34:525–533.PubMedCrossRefGoogle Scholar
  4. Borisy, G. G., and Taylor, E. W., 1967b, The mechanism of action of colchicine: II. Colchicine binding to sea urchin eggs and the mitotic apparatus, J. Cell Biol. 34:535–548.PubMedCrossRefGoogle Scholar
  5. Carter, S. B., 1967, Effects of cytochalasins on mammalian cells, Nature 213:261–264.PubMedCrossRefGoogle Scholar
  6. Cheung, H. T., Cantarow, W. D., and Sundhasadas, G., 1978, Colchicine and cytochalasin B effects on random movement, spreading and adhesion of mouse macrophages, Exp. Cell Res. 111:95–103.PubMedCrossRefGoogle Scholar
  7. Clark, M. A., Crenshaw, A. H., and Shay, J. W., 1978, Fusion of mammalian somatic cells with polyethylene glycol 400 MW, Tissue Culture Assoc. Manual 4:801–804.CrossRefGoogle Scholar
  8. Cremer, T., Zorn, C., and Zimmer, J., 1976, Formation of viable cell fragments by treatment with colchicine, Exp. Cell Res. 100:345–355.PubMedCrossRefGoogle Scholar
  9. Crenshaw, A. H., and Murrell, L. R., 1980, Micronucleation of human fibroblasts with Colcemid, In Vitro 16:257–258.Google Scholar
  10. Crenshaw, A. H., Shay, J. W., and Murrell, L. R., 1980, Mass enucleation of tissue culture cell monolayers. J. Tissue Culture Methods 6:127–130.CrossRefGoogle Scholar
  11. Crenshaw, A. H., Shay, J. W., and Murrell, L. R., 1981a, Colcemid-induced micronucleation in cultured human cells, J. Ultrastruct. Res. 75:179–186.PubMedCrossRefGoogle Scholar
  12. Crenshaw, A. H., Shay, J. W., and Murrell, L. R., 1981b, Micronucleation of human somatic cells with Colcemid, J. Tissue Culture Methods (in press).Google Scholar
  13. Croop, J., and Holtzer, H., 1975, Response of myogenic and fibrogenic cells to cytochalasin B and Colcemid: I. Light microscopic observations, J. Cell Biol. 65:271–285.PubMedCrossRefGoogle Scholar
  14. Davidson, R. L., and Gerald, P. S., 1977, Induction of mammalian somatic cell hybridization by polyethylene glycol, in: Methods in Cell Biology, Volume XV (D. M. Prescott, ed.), Academic Press, New York, pp. 325–338.Google Scholar
  15. Ege, T., and Ringertz, N. R., 1974, Preparation of microcells by enucleation of micronucleate cells, Exp. Cell Res. 87:378–382.PubMedCrossRefGoogle Scholar
  16. Ege, T., Krondahl, U., and Ringertz, N. R., 1974, Introduction of nuclei and micronuclei into cells and enucleated cytoplasms by Sendai virus induced fusion, Exp. Cell Res. 88:428–432.PubMedCrossRefGoogle Scholar
  17. Ege, T., Ringertz, N. R., Hamberg, H., and Sidebottom, E., 1977, Preparation of microcells, in: Methods in Cell Biology, Volume XV (D. M. Prescott, ed.), Academic Press, New York, pp. 339–357.Google Scholar
  18. Fournier, R. E. K., and Ruddle, F. H., 1977, Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells, Proc. Natl. Acad. Sci. USA 74:319–323.PubMedCrossRefGoogle Scholar
  19. Harris, H., and Watkins, J. F., 1965, Hybrid cells derived from mouse and man: Artificial heterokaryons of mammalian cells from different species, Nature 205:640.PubMedCrossRefGoogle Scholar
  20. Johnson, R.T., Mulliger, A. M., and Skaer, R. J., 1975, Perturbation of mammalian cell division: I. Human mini segregants derived from mitotic cells, Proc. R. Soc. Lond. B 189:591–602.PubMedCrossRefGoogle Scholar
  21. Johnson, R. T., Mullinger, A. M., and Downes, C. S., 1978, Human minisegregant cells, in: Methods in Cell Biology, Volume XX (D. M. Prescott, ed.), Academic Press, New York, pp. 255–315.Google Scholar
  22. Littlefield, J. W., 1966, The use of drug-resistant markers to study the hybridization of mouse fibroblasts, Exp. Cell Res. 41:190–196.PubMedCrossRefGoogle Scholar
  23. Lucas, J. J., Szekely, E., and Kates, J. R., 1976, The construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation, Cell 7:397–405.PubMedCrossRefGoogle Scholar
  24. Margulis, L., 1973, Colchicine-sensitive microtubules, Int. Rev. Cytol. 34:333–361.PubMedCrossRefGoogle Scholar
  25. McBride, O. W., and Ozer, H. L., 1973, Transfer of genetic information by purified metaphase chromosomes, Proc. Natl. Acad. Sci. USA 70:1258–1262.PubMedCrossRefGoogle Scholar
  26. McKusick, V. A., and Ruddle, F. H., 1977, The status of the gene map of the human chromosomes, Science 196:309–405.CrossRefGoogle Scholar
  27. McNeill, C. A., and Brown, R. L., 1980, Genetic manipulation by means of microcell-mediated transfer of normal human chromosomes into recipient mouse cells, Proc. Natl. Acad. Sci. USA 77:5394–5398.PubMedCrossRefGoogle Scholar
  28. Mullinger, A. M., and Johnson, R. T., 1976, Perturbation of mammalian cell division: III. The topology and kinetics of extrusion subdivision, J. Cell Sci. 22:243–285.PubMedGoogle Scholar
  29. Peterson, D. F., Anderson, E. C., and Tobey, R. A., 1968, Mitotic cells as a source of synchronized cultures, in: Methods in Cell Physiology, Volume III (D. M. Prescott, ed.), Academic Press, New York, pp. 347–370.Google Scholar
  30. Phillips, S. G., and Phillips, D. M., 1969, Sites of nucleolus production in cultured Chinese hamster cells, J. Cell Biol. 40:248–268.PubMedCrossRefGoogle Scholar
  31. Pontecorvo, G., 1975, Production of independently multiplying mammalian somatic cell hybrids by polyethylene glycol (PEG) treatment, Somat. Cell Genet. 1:397–400.PubMedCrossRefGoogle Scholar
  32. Poste, G., 1973, Anucleate mammalian cells: Applications in cell biology and virology, in: Methods in Cell Biology, Volume VII (D. M. Prescott, ed.), Academic Press, New York, pp. 211–249.Google Scholar
  33. Prescott, D. M., and Kirkpatrick, J. B., 1973, Mass enucleation of cultured animal cells, in: Methods in Cell Biology, Volume VII (D. M. Prescott, ed.), Academic Press, New York, pp. 189–202.Google Scholar
  34. Prescott, D. M., Myerson, D., and Wallace, J., 1972, Enucleation of mammalian cells with cytochalasin B, Exp. Cell Res. 71:480–485.PubMedCrossRefGoogle Scholar
  35. Ruddle, F. H., and Creagan, R. P., 1975, Parasexual approaches to the genetics of man, Ann. Rev. Genet. 9:407–486.PubMedCrossRefGoogle Scholar
  36. Schor, S. L., Johnson, R. T., and Mullinger, M. A., 1975, Perturbation of mammalian cell division: II. Studies on the isolation and characterization of human mini segregant cells, J. Cell Sci. 19:281–303.PubMedGoogle Scholar
  37. Shay, J. W., and Clark, M. A., 1977, Morphological studies on the enucleation of colchicine treated L-929 cells, J. Ultrastruct. Res. 58:155–159.CrossRefGoogle Scholar
  38. Shay, J. W., Porter, K. R., and Prescott, D. M., 1973, The surface morphology and fine structure of CHO (Chinese hamster ovary) cells following enucleation, Proc. Natl. Acad. Sci. USA 71:3059–3063.CrossRefGoogle Scholar
  39. Subblefield, E., 1964, DNA synthesis and chromosome morphology of Chinese hamster cells cultured in media containing N-desacyl-N-methyl-colchicine (Colcemid), in: Cytogenetics of Cells in Culture, Volume 3 (R. J. C. Harris, ed.), Academic Press, New York, pp. 223–298.Google Scholar
  40. Sundar Raj, C. V., Church, R. L., Klobutcher, L. A., and Ruddle, F. H., 1977, Genetics of the connective tissue proteins: Assignment of the gene of human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids, Proc. Natl. Acad. Sci. USA 74:4444–4448.PubMedCrossRefGoogle Scholar
  41. Tourian, A., Johnson, R. T., Burg, K., Nicholson, S. W., and Sperling, K., 1978, Transfer of human chromosomes via human mini segregant cells into mouse cells and the quantitation of the expression of hypoxanthine phosphoribosyl-transferase in the hybrids, J. Cell Sci. 30:193–209.PubMedGoogle Scholar
  42. Veomett, G., Shay, J. W., Hough, P. V. C, and Prescott, D. M., 1976, Large scale enucleation of mammalian cells, in: Methods in Cell Biology, Volume XIII (D. M. Prescott, ed.), Academic Press, New York, pp. 1–3.Google Scholar
  43. Weiss, M. C, and Green, H., 1967, Human-mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes, Proc. Natl. Acad. Sci. USA 58:1104–1111.PubMedCrossRefGoogle Scholar
  44. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M., 1971, Microfilaments in cellular and developmental processes: Contractile microfilament machinery of many cell types is reversibly inhibited by cytochalasin B, Science 171:135–143.PubMedCrossRefGoogle Scholar
  45. Willecke, K., and Ruddle, F. H., 1975, Transfer of the human gene for hypoxanthine-guanine phosphoribosyltransferase via isolated metaphase chromosomes into mouse L-cells, Proc. Natl. Acad. Sci. USA 72:1792–1796.PubMedCrossRefGoogle Scholar
  46. Wright, W. E., 1973, The production of mass populations of anucleate cytoplasms, in: Methods in Cell Biology, Volume VII (D. M. Prescott, ed.), Academic Press, New York, pp. 203–210.Google Scholar
  47. Wright, W. E., and Hayflick, L., 1972, Formation of anucleate and multinucleate cells in normal and SV40 transformed WI-38 by cytochalasin B, Exp. Cell Res. 74:187–194.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1982

Authors and Affiliations

  • Andrew H. CrenshawJr.
    • 1
  • Leonard R. Murrell
    • 1
  1. 1.Department of AnatomyUniversity of Tennessee Center for the Health SciencesMemphisUSA

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