Evaluation of Extrachromosomal Gene Copy Number of Transiently Transfected Cell Lines
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DNA that is taken up, remains circular and extrachromosomal, and is present as chromatin (8,9), and Wilson and Patient, unpublished).
KeywordsTransfection Protocol Carrier Substance Thick Boundary Layer Plasmid Dilution Test Plasmid
- 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.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
- 7.Wilson, A. C. and Patient, R. K. Scrapefection-A rapid and simple method for transfecting adherent cells. Manuscript in prep.Google Scholar
- 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
- 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