Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745

High-efficiency transformation of mammalian cells by plasmid DNA.

C Chen 1, H Okayama 1
PMCID: PMC367891  PMID: 3670292

Abstract

We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.

Full text

PDF

Images in this article

2747Image
on p.2747
2747Image
on p.2747
2748Image
on p.2748
2751Image
on p.2751

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banerji J., Olson L., Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell. 1983 Jul;33(3):729–740. doi: 10.1016/0092-8674(83)90015-6. [DOI] [PubMed] [Google Scholar]
  2. Benoist C., Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981 Mar 26;290(5804):304–310. doi: 10.1038/290304a0. [DOI] [PubMed] [Google Scholar]
  3. Bouck N., di Mayorca G. Somatic mutation as the basis for malignant transformation of BHK cells by chemical carcinogens. Nature. 1976 Dec 23;264(5588):722–727. doi: 10.1038/264722a0. [DOI] [PubMed] [Google Scholar]
  4. Breitman M. L., Tsui L. C., Buchwald M., Siminovitch L. Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells. Mol Cell Biol. 1982 Aug;2(8):966–976. doi: 10.1128/mcb.2.8.966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Corsaro C. M., Pearson M. L. Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells. Somatic Cell Genet. 1981 Sep;7(5):603–616. doi: 10.1007/BF01549662. [DOI] [PubMed] [Google Scholar]
  6. Fromm M., Berg P. Deletion mapping of DNA regions required for SV40 early region promoter function in vivo. J Mol Appl Genet. 1982;1(5):457–481. [PubMed] [Google Scholar]
  7. Ghosh P. K., Lebowitz P., Frisque R. J., Gluzman Y. Identification of a promoter component involved in positioning the 5' termini of simian virus 40 early mRNAs. Proc Natl Acad Sci U S A. 1981 Jan;78(1):100–104. doi: 10.1073/pnas.78.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gorman C., Padmanabhan R., Howard B. H. High efficiency DNA-mediated transformation of primate cells. Science. 1983 Aug 5;221(4610):551–553. doi: 10.1126/science.6306768. [DOI] [PubMed] [Google Scholar]
  9. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  10. Ishiura M., Hirose S., Uchida T., Hamada Y., Suzuki Y., Okada Y. Phage particle-mediated gene transfer to cultured mammalian cells. Mol Cell Biol. 1982 Jun;2(6):607–616. doi: 10.1128/mcb.2.6.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jolly D. J., Esty A. C., Bernard H. U., Friedmann T. Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1982 Aug;79(16):5038–5041. doi: 10.1073/pnas.79.16.5038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jolly D. J., Okayama H., Berg P., Esty A. C., Filpula D., Bohlen P., Johnson G. G., Shively J. E., Hunkapillar T., Friedmann T. Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase. Proc Natl Acad Sci U S A. 1983 Jan;80(2):477–481. doi: 10.1073/pnas.80.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  15. Kühn L. C., McClelland A., Ruddle F. H. Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell. 1984 May;37(1):95–103. doi: 10.1016/0092-8674(84)90304-0. [DOI] [PubMed] [Google Scholar]
  16. Lewis W. H., Srinivasan P. R., Stokoe N., Siminovitch L. Parameters governing the transfer of the genes for thymidine kinase and dihydrofolate reductase into mouse cells using metaphase chromosomes or DNA. Somatic Cell Genet. 1980 May;6(3):333–347. doi: 10.1007/BF01542787. [DOI] [PubMed] [Google Scholar]
  17. Littlefield J. W. The use of drug-resistant markers to study the hybridization of mouse fibroblasts. Exp Cell Res. 1966 Jan;41(1):190–196. doi: 10.1016/0014-4827(66)90558-1. [DOI] [PubMed] [Google Scholar]
  18. Lowy D. R., Rands E., Scolnick E. M. Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J Virol. 1978 May;26(2):291–298. doi: 10.1128/jvi.26.2.291-298.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luthman H., Magnusson G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 1983 Mar 11;11(5):1295–1308. doi: 10.1093/nar/11.5.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  21. McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  22. Neumann E., Schaefer-Ridder M., Wang Y., Hofschneider P. H. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1982;1(7):841–845. doi: 10.1002/j.1460-2075.1982.tb01257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okayama H., Berg P. Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells. Mol Cell Biol. 1985 May;5(5):1136–1142. doi: 10.1128/mcb.5.5.1136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Perucho M., Hanahan D., Lipsich L., Wigler M. Isolation of the chicken thymidine kinase gene by plasmid rescue. Nature. 1980 May 22;285(5762):207–210. doi: 10.1038/285207a0. [DOI] [PubMed] [Google Scholar]
  28. Queen C., Baltimore D. Immunoglobulin gene transcription is activated by downstream sequence elements. Cell. 1983 Jul;33(3):741–748. doi: 10.1016/0092-8674(83)90016-8. [DOI] [PubMed] [Google Scholar]
  29. Schechter A. L., Stern D. F., Vaidyanathan L., Decker S. J., Drebin J. A., Greene M. I., Weinberg R. A. The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature. 1984 Dec 6;312(5994):513–516. doi: 10.1038/312513a0. [DOI] [PubMed] [Google Scholar]
  30. Schöler H. R., Gruss P. Specific interaction between enhancer-containing molecules and cellular components. Cell. 1984 Feb;36(2):403–411. doi: 10.1016/0092-8674(84)90233-2. [DOI] [PubMed] [Google Scholar]
  31. Shih C., Shilo B. Z., Goldfarb M. P., Dannenberg A., Weinberg R. A. Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5714–5718. doi: 10.1073/pnas.76.11.5714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shih C., Weinberg R. A. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell. 1982 May;29(1):161–169. doi: 10.1016/0092-8674(82)90100-3. [DOI] [PubMed] [Google Scholar]
  33. Shimizu K., Goldfarb M., Perucho M., Wigler M. Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line. Proc Natl Acad Sci U S A. 1983 Jan;80(2):383–387. doi: 10.1073/pnas.80.2.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  35. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  36. Takeishi K., Ayusawa D., Kaneda S., Shimizu K., Seno T. Molecular cloning of genomic DNA segments partially coding for human thymidylate synthase from the mouse cell transformant. J Biochem. 1984 May;95(5):1477–1483. doi: 10.1093/oxfordjournals.jbchem.a134755. [DOI] [PubMed] [Google Scholar]
  37. Westerveld A., Hoeijmakers J. H., van Duin M., de Wit J., Odijk H., Pastink A., Wood R. D., Bootsma D. Molecular cloning of a human DNA repair gene. Nature. 1984 Aug 2;310(5976):425–429. doi: 10.1038/310425a0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES