Techniques for Purifying L-Cell Karyoplasts with Minimal Amounts of Cytoplasm

  • Mike A. Clark
  • Jerry W. Shay


Cells may be enucleated by centrifuging monolayers of cells in the presence of the drug cytochalasin B (CB) (Prescott et al., 1972; Wright and Hayflick, 1972). These procedures allow one to obtain large numbers of enucleated cells (cytoplasts) and nuclei surrounded by a thin layer of cytoplasm containing an intact outer plasma membrane. The nuclear fragments were originally termed karyoplasts and described as being incapable of regeneration. Later it was reported that cytoplasts could be fused to karyoplasts using inactivated Sendai virus (Veomett et al., 1974). The resulting “reconstructed” cells are of potential use in many aspects of somatic cell studies in areas such as gene regulation, differentiation, and transformation (Shay, 1977; Shay and Clark 1979) (also see Chapter 19, this volume). A major difficulty in these experimental procedures has been the presence of a low percentage of cells in the cytoplast population which fail to enucleate and the more serious problem of a small fraction (approximately 6%) of the whole cells which detach during enucleation and thus contaminate the karyoplast population.


Ficoll Gradient Nuclear Donor Separation Chamber Differential Adhesion Exclude Trypan Blue 
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  1. Clark, M. A., Crenshaw, A. H., and Shay, J. W., 1977, Fusion of mammalian somatic cells with polyethylene glycol 400 M.W., Tissue Culture Assoc. Manual 4(2):801–804.CrossRefGoogle Scholar
  2. Crissman, H. A., and Tobey, R. A., 1974, Cell cycle analysis in 20 minutes, Science 184:1297–1298.PubMedCrossRefGoogle Scholar
  3. Hayflick, L., 1965, Tissue cultures and mycoplasmas, Texas Rep. Biol. Med. 23:285–303.Google Scholar
  4. Lucas, J. J., Szakely, E., and Kates, J. R., 1976, The regeneration and division of mouse L-cell karyoplasts, Cell 7:115–130.PubMedCrossRefGoogle Scholar
  5. Prescott, D. M., Myerson, D., and Wallace, J., 1972, Enucleation of mammalian cells with cytochalasin B, Exp. Cell Res. 71:480–485.PubMedCrossRefGoogle Scholar
  6. Schneider, E. L., Stanbridge, E. J., and Epstein, C. J., 1974, Incorporation of 3H-uridine and 3H-uracil into RNA, Exp. Cell Res. 84:311–318.PubMedCrossRefGoogle Scholar
  7. Shall, S., 1975, Selection synchronization by velocity sedimentation separation of mouse fibroblasts, in: Methods in Ceil Biology, Volume VII (D. M. Prescott, ed.), Academic Press, New York, pp. 269–285.Google Scholar
  8. Shay, J. W., 1977, Selection of reconstituted cells from karyoplasts fused to chloramphenicol resistant cytoplasts, Proc. Natl. Acad. Sci. USA 74:2461–2464.PubMedCrossRefGoogle Scholar
  9. Shay, J. W., and Clark, M. A., 1977, Morphological studies on the enucleation of colchicine treated L-929 cells, J. Ultrastruct. Res. 58:155–161.CrossRefGoogle Scholar
  10. Shay, J. W., and Clark, M. A., 1979, Nuclear control of tumorigenicity in cells reconstructed by PEG induced fusion of cell fragments, J. Supermol Struct. 11:33–49.CrossRefGoogle Scholar
  11. Tai, Y. H., and Quinn, P. A., 1977, Rapid detection of mycoplasma contamination in tissue cultures by SEM, Scanning Electron Microscopy 2:291–299.Google Scholar
  12. Veomett, G., Prescott, D. M., Shay, J., andPorter, K. R., 1974, Reconstruction of mammalian cells from nucleic and cytoplasmic components separated by treatment with cytochalasin B, Proc. Natl. Acad. Sci. USA 71:1999–2002.PubMedCrossRefGoogle Scholar
  13. Wright, W. E., and Hayflick, L., 1972, Formation of anucleate cells in normal and SV40 transformed WI-38 by cytochalasin B, Exp. Cell Res. 74:187–194.PubMedCrossRefGoogle Scholar
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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
  21. 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
  22. 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
  23. 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

  • Mike A. Clark
    • 1
  • Jerry W. Shay
    • 1
  1. 1.Department of Cell BiologyUniversity of Texas Health Science CenterDallasUSA

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