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Nucleic Acids Res. Feb 15, 1996; 24(4): 596–601.
PMCID: PMC145683

Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation.


DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes. The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as well. An example for this is the finding that almost all of the soluble DNA in the reaction mix can be bound into an insoluble complex with calcium phosphate in <1 min. Extending the reaction time to 20 min results in aggregation and/or growth of particles and reduces the level of expression. With improved protocols we gained better reproducibility and higher efficiencies both for transient and for stable transfections. Up to 60% of cells stained positive for beta-gal and transient production of secreted proteins was improved 5- to 10-fold over results seen with transfections using standard procedures. Similar improvements in efficiency (number of recombinant cell colonies) were observed with stable transfections, using co-transfected marker plasmids for selection. Transient expression levels 2 days after DNA transfer and titers obtained from stable cell lines, emerging weeks later, showed strong correlation.

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Selected References

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  • Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. [PubMed]
  • Loyter A, Scangos GA, Ruddle FH. Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci U S A. 1982 Jan;79(2):422–426. [PMC free article] [PubMed]
  • Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. [PMC free article] [PubMed]
  • Kjer KM, Fallon AM. Efficient transfection of mosquito cells is influenced by the temperature at which DNA-calcium phosphate coprecipitates are prepared. Arch Insect Biochem Physiol. 1991;16(3):189–200. [PubMed]
  • O'Mahoney JV, Adams TE. Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection. DNA Cell Biol. 1994 Dec;13(12):1227–1232. [PubMed]
  • Paborsky LR, Fendly BM, Fisher KL, Lawn RM, Marks BJ, McCray G, Tate KM, Vehar GA, Gorman CM. Mammalian cell transient expression of tissue factor for the production of antigen. Protein Eng. 1990 May;3(6):547–553. [PubMed]
  • Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. [PMC free article] [PubMed]
  • Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. [PMC free article] [PubMed]
  • Ringold G, Dieckmann B, Lee F. Co-expression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells. J Mol Appl Genet. 1981;1(3):165–175. [PubMed]
  • Kaufman RJ, Sharp PA. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. J Mol Biol. 1982 Aug 25;159(4):601–621. [PubMed]
  • Wurm FM. Integration, amplification and stability of plasmid sequences in CHO cell cultures. Biologicals. 1990 Jul;18(3):159–164. [PubMed]
  • MacGregor GR, Caskey CT. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 1989 Mar 25;17(6):2365–2365. [PMC free article] [PubMed]
  • Paoni NF, Keyt BA, Refino CJ, Chow AM, Nguyen HV, Berleau LT, Badillo J, Peña LC, Brady K, Wurm FM, et al. A slow clearing, fibrin-specific, PAI-1 resistant variant of t-PA (T103N, KHRR 296-299 AAAA). Thromb Haemost. 1993 Aug 2;70(2):307–312. [PubMed]
  • Keyt BA, Paoni NF, Refino CJ, Berleau L, Nguyen H, Chow A, Lai J, Peña L, Pater C, Ogez J, et al. A faster-acting and more potent form of tissue plasminogen activator. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3670–3674. [PMC free article] [PubMed]
  • Brash DE, Reddel RR, Quanrud M, Yang K, Farrell MP, Harris CC. Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells. Mol Cell Biol. 1987 May;7(5):2031–2034. [PMC free article] [PubMed]
  • Sanes JR, Rubenstein JL, Nicolas JF. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. [PMC free article] [PubMed]

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