Advertisement

Calcium Phosphate Transfection of Mammalian Cultured Cells

  • Elaine T. Schenborn
  • Virginia Goiffon
Part of the Methods in Molecular Biology™ book series (MIMB, volume 130)

Abstract

Calcium phosphate and DEAE-dextran reagents were incorporated into the first chemical methods that successfully transferred nucleic acid directly to cultured mammalian cells in a process referred to as transfection (1, 2, 3, 4). Early transfection studies used viral RNA (1) and DNA (2,4), which, at that time, were relatively easy to propagate and purify, and allowed phenotypic discrimination of the transfected mammalian cells. Calcium phosphate coprecipitation and DEAE-dextran methods became widely used after cloning and manipulation of plasmid DNA became routine, and it was demonstrated that plasmid DNA was effectively transferred to cultured cells using these methods. Together with advancements in vector development came the introduction of additional transfection methods using chemical reagents such as liposomes (5,6), dendrimers (7,8), and cationic polymers (9,10), plus physical methods such as electroporation (11) and biolistic microparticle bombardment (12). However, the calcium phosphate coprecipitation technique still remains one of the most widely used in vitro transfection methods.

Keywords

Calcium Phosphate Stable Transfection Fresh Growth Medium Shock Solution Neomycin Phosphotransferase Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Vaheri, A. and Pagano, J.S. (1965) Infectious poliovirus RNA: a sensitive method of assay. Virology 27, 434–436.PubMedCrossRefGoogle Scholar
  2. 2.
    McCutchan, J. H. and Pagano, J. S. (1968) Enhancement of the infectivity of Simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Natl. Cancer Inst. 41, 351–357.PubMedGoogle Scholar
  3. 3.
    Szybalska, E. H. and Szybalski, W. (1962) Genetics of human cell lines IV. DNA-mediated heritable transformation of a biochemical trait. Proc. Natl. Acad. Sci. USA 48, 2026–2034.PubMedCrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    Fraley, R., Subramani, S., Berg, P., and Papahadjopoulos, D. (1980) Introduction of liposome-encapsulated SV40 DNA into cells. J. Biol. Chem. 255, 10,431–10,435.PubMedGoogle Scholar
  6. 6.
    Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection: A highly efficient, lipid mediated DNA transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413–7417.PubMedCrossRefGoogle Scholar
  7. 7.
    Haensler, J. and Szoka, F. C. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconj. Chem. 4, 372–379.CrossRefGoogle Scholar
  8. 8.
    Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Spindler, R., Tomalia, D. A., and Baker, J. R. (1996) Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. Proc. Natl. Acad. Sci. USA 93, 4897–4902.PubMedCrossRefGoogle Scholar
  9. 9.
    Farber, R. E., Melnick, J. L., and Butel, J. S. (1975) Optimal conditions for uptake of exogenous DNA by chinese hamster lung cells deficient in hypoxanthine-guanosine phosphoribosyl transferase. Biochim. Biophys. Acta 390, 298–311.PubMedGoogle Scholar
  10. 10.
    Boussif, O., Lezoualch, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92, 7297–7301.PubMedCrossRefGoogle Scholar
  11. 11.
    Wong, T. K. and Neumann, E. (1982) Electric field mediated gene transfer. iochem. Biophys. Res. Commun. 107, 584–587.CrossRefGoogle Scholar
  12. 12.
    Ye, G. N., Danielle, H., and Sanford, J. C. (1990) Optimization of delivery of foreign DNA into higher-plant chloroplasts. Plant Molec. Biol. 15, 809–819.CrossRefGoogle Scholar
  13. 13.
    Wigler, M., Silverstein, S., Lee, L. S., Pellicer, A., Cheng, Y., and Axel R. (1977) Transfer of purified Herpes Virus thymidine kinase gene to cultured mouse cells. Cell 11, 223–232.PubMedCrossRefGoogle Scholar
  14. 14.
    Loyter, A., Scangos, G. A., and Ruddle, F. H. (1982) Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc. Natl. Acad. Sci. USA 79, 422–426.PubMedCrossRefGoogle Scholar
  15. 15.
    Chu, G. and Sharp, P. A. (1981) SV40 DNA transfection of cells in suspension: analysis of efficiency of transcription and translation of T antigen. Gene 13, 197–202.PubMedCrossRefGoogle Scholar
  16. 16.
    Jordan, M., Schallhorn, A., and Wurm, F. M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucl. Acids Res. 24, 596–601.PubMedCrossRefGoogle Scholar
  17. 17.
    Chen, C. and Okayama, H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752.PubMedGoogle Scholar
  18. 18.
    Chen, C. A. and Okayama, H. (1988) Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6, 632–638.PubMedGoogle Scholar
  19. 19.
    Kingston, R. E. (1995) Introduction of DNA into mammalian cells, in Current Protocols in Molecular Biology (Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G. Smith, J. A., and Struhl, K., eds.), Wiley, New York.Google Scholar
  20. 20.
    Jakoby, W. B. and Pastan, I. H., eds. (1979) Methods in Enzymology 58, Academic, New York, NY.Google Scholar
  21. 21.
    Frost, E. and Williams, J. (1978) Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology 91, 39–50.PubMedCrossRefGoogle Scholar
  22. 22.
    Wilson, S. P., Liu, F. L., Wilson, R. E., and Housley, P. R. (1995) Optimization of calcium phosphate transfection for bovine chromaffin cells: relationship to calcium phosphate precipitate formation. Anal. Biochem. 226, 212–220.PubMedCrossRefGoogle Scholar
  23. 23.
    Zauner, W., Kichler, A., Schmidt, W., Sinski, A., and Wagner, E. (1996) Glycerol enhancement of ligand-polylysine / DNA transfection. Biotechniques 20, 905–913.PubMedGoogle Scholar
  24. 24.
    Lowy, D. R., Rands, E., and Scolnick, E. M. (1978) Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J. Virology 26, 291–298.PubMedGoogle Scholar
  25. 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. 26.
    Sambrook, J., Fritsch, E. F., and Maniatis, T., ed (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  27. 27.
    Freshney, R. I., ed. (1986) Animal Cell Culture. IRL Press, Oxford, England, pp. 1–11.Google Scholar
  28. 28.
    Freshney, R. I., ed. (1987) Culture of Animal Cells. A.R. Liss, Inc., New York, pp. 51–59.Google Scholar
  29. 29.
    Seelos, C. (1997) A critical parameter determining the aging of DNA-calcium phosphate precipitates. Anal. Biochem. 245, 109–111.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Elaine T. Schenborn
    • 1
  • Virginia Goiffon
    • 1
  1. 1.Promega CorporationMadison

Personalised recommendations