Evaluation of Extrachromosomal Gene Copy Number of Transiently Transfected Cell Lines

  • Angus C. Wilson
  • Roger K. Patient
Part of the Methods in Molecular Biology book series (MIMB, volume 7)

Abstract

The short-term expression of DNA introduced into eukaryotic cells is now widely used to investigate the biological activities of cellular and viral genes or their products. A number of different transfection methods are in common use and can be broadly divided into two categories, based on the method by which DNA is introduced (either as a complex with a carrier substance that facilitates uptake by the cell [e.g., DEAE-dextran transfection (1,2), calcium phosphate coprecipitation (3), or lipofection (4) or by direct exposure to the cytoplasm [e.g., electroporation (5), microinjection (6) or scrapefection (7). In each case, transfected cells can maintain a considerable number of plasmids in their nuclei for several cell cycles. Plasmids lacking functional replication origins are lost progressively in subsequent mitoses. It appears that, for at least some transfection protocols, the majority of DNA that is taken up, remains circular and extrachromosomal, and is present as chromatin (8,9), and Wilson and Patient, unpublished).

Keywords

Vortex Glycerol Phenol Carbohydrate Agarose 

References

  1. 1.
    McCutchan, J. H. and Pagano, J. S. (1968) Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethyl-amino-ethyldextran. J Natl. Cancer Inst. 41, 351–356.PubMedGoogle Scholar
  2. 2.
    Lopata, M., Cleveland, D., and Sollner-Webb, B. (1984) High level transient expression of chloramphenicol acetyl transferase gene by DEAEdextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nuclcc Acids Res. 2, 5707–5717.CrossRefGoogle Scholar
  3. 3.
    Graham, F. L. and van der Eb, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467.PubMedCrossRefGoogle Scholar
  4. 4.
    Fraley, R., Subramani, P., Berg, P., and Papahadjopoulos, P. (1980) Introduction of liposomeencapsulated SV40 DNA into cells. J Riol. Chem. 255, 10431–10435.Google Scholar
  5. 5.
    Andreason, G. L. and Evans, G. A. (1988) Introduction and expression of DNA molecules in eukaryotic cells by electroporation. BioTechniqucs 6, 650–660.Google Scholar
  6. 6.
    Capecchi, M. R. (1980) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. cell 22, 479–488.PubMedCrossRefGoogle Scholar
  7. 7.
    Wilson, A. C. and Patient, R. K. Scrapefection-A rapid and simple method for transfecting adherent cells. Manuscript in prep.Google Scholar
  8. 8.
    Enver, T., Brewer, A. C., and Patient, R. K. (1985) Simian virus 40-mediated cis induction of theXenopus p globin DNase I hypersensitive site. Nature 318, 680–683.PubMedCrossRefGoogle Scholar
  9. 9.
    Brewer, A. C., Enver, T., Greaves, D. R., Allan, J., and Patient, R. R. (1988) 5’ structural motifs andXenopus globin gene activation. J Mol. Biol. 199, 535–585.CrossRefGoogle Scholar
  10. 10.
    Hirt, B. (1967) Selective extraction of polyoma DNA from transfected mouse cell cultures. j Mol. Biol 26, 365–369.PubMedCrossRefGoogle Scholar
  11. 11.
    Ogden, R. C. and Adams, D. A. (1987) Electrophoresis in agarose and acrylamide gels, in Methods in Enzymology, vol. 152 (Berger, S. L. and Kimmel, A. R., eds., Academic, New York, London, 61–90.Google Scholar
  12. 12.
    Wahl, G. M., Meinkoth, J. L., and Rimmel, A. R. (1987) Northern and Southern Blots,in Methods in Enzymology, vol. 152 (edBerger, S. L. and Kimmel, A. R., eds., Academic New York, London, 61–90.CrossRefGoogle Scholar
  13. 13.
    Lehman, A. R. and Oomen, A. (1985) Effect of DNA damage on the expression of CAT after transfection. Nucleic Acids Res. 18, 2087–2095.CrossRefGoogle Scholar
  14. 14.
    Weintraub, H., Cheng, P. F., and Conrad, K (1986) Expression of transfected DNA depends on DNA topology. Cell 46, 115–122.PubMedCrossRefGoogle Scholar
  15. 15.
    Shure, M., Pulleybank, D. E., and Vinograd, J. (1977) The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix densiv heterogeneity. Nucleic Acids Res. 4, 1183–1205.PubMedCrossRefGoogle Scholar
  16. 16.
    Alwine, J. C. (1985) Transient gene expression control: Effects of transfected DNA stability and truwactivation by viral early proteins. Mol. cell. Biol. 5, 1034–1042.PubMedGoogle Scholar
  17. 17.
    Laskey, R. A. and Mills, A. D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur.J Eiuchem. 56, 335–341.Google Scholar

Copyright information

© The Humana Press Inc., Clifton, NJ 1991

Authors and Affiliations

  • Angus C. Wilson
    • 1
  • Roger K. Patient
    • 1
  1. 1.Kind’s CollegeUniversity of LondonLondonUK

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