Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 May 25;17(10):3959–3971. doi: 10.1093/nar/17.10.3959

Enhanced transfection efficiency and improved cell survival after electroporation of G2/M-synchronized cells and treatment with sodium butyrate.

S Goldstein 1, C M Fordis 1, B H Howard 1
PMCID: PMC317872  PMID: 2786626

Abstract

To achieve high transfection efficiency in human fibroblasts with good preservation of proliferative capacity we developed an electroporation procedure that combines two distinct modalities: use of recipient cells synchronized in the late G2/mitotic phase of the cell cycle and treatment of cells post-electroporation with 5 mM butyrate. This combination enabled reduction of plasmid DNA concentration and electroporation voltage, both associated with cytotoxicity, while greatly enhancing transfection efficiencies. Although the method was primarily developed for transient expression it was also found to improve stable expression. This procedure should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.

Full text

PDF
3959

Images in this article

3964Image
on p.3964

Selected References

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

  1. Brash D. E., Reddel R. R., Quanrud M., Yang K., Farrell M. P., Harris C. C. 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. doi: 10.1128/mcb.7.5.2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Candido E. P., Reeves R., Davie J. R. Sodium butyrate inhibits histone deacetylation in cultured cells. Cell. 1978 May;14(1):105–113. doi: 10.1016/0092-8674(78)90305-7. [DOI] [PubMed] [Google Scholar]
  3. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu G., Hayakawa H., Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987 Feb 11;15(3):1311–1326. doi: 10.1093/nar/15.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cone R. D., Mulligan R. C. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6349–6353. doi: 10.1073/pnas.81.20.6349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fordis C. M., Howard B. H. Use of the CAT reporter gene for optimization of gene transfer into eukaryotic cells. Methods Enzymol. 1987;151:382–397. doi: 10.1016/s0076-6879(87)51030-8. [DOI] [PubMed] [Google Scholar]
  7. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  8. Gorman C. M., Howard B. H., Reeves R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 1983 Nov 11;11(21):7631–7648. doi: 10.1093/nar/11.21.7631. [DOI] [PMC free article] [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. Gupta R. S., Goldstein S. Diphtheria toxin resistance in human fibroblast cell strains from normal and cancer-prone individuals. Mutat Res. 1980 Dec;73(2):331–338. doi: 10.1016/0027-5107(80)90198-0. [DOI] [PubMed] [Google Scholar]
  11. Hecht F., Glover T. W. Cancer chromosome breakpoints and common fragile sites induced by aphidicolin. Cancer Genet Cytogenet. 1984 Oct;13(2):185–188. doi: 10.1016/0165-4608(84)90060-8. [DOI] [PubMed] [Google Scholar]
  12. Kinosita K., Jr, Tsong T. Y. Voltage-induced pore formation and hemolysis of human erythrocytes. Biochim Biophys Acta. 1977 Dec 1;471(2):227–242. doi: 10.1016/0005-2736(77)90252-8. [DOI] [PubMed] [Google Scholar]
  13. Kruh J. Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Mol Cell Biochem. 1982 Feb 5;42(2):65–82. doi: 10.1007/BF00222695. [DOI] [PubMed] [Google Scholar]
  14. Kurata S., Tsukakoshi M., Kasuya T., Ikawa Y. The laser method for efficient introduction of foreign DNA into cultured cells. Exp Cell Res. 1986 Feb;162(2):372–378. doi: 10.1016/0014-4827(86)90342-3. [DOI] [PubMed] [Google Scholar]
  15. Liu P. K., Chang C. C., Trosko J. E., Dube D. K., Martin G. M., Loeb L. A. Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha. Proc Natl Acad Sci U S A. 1983 Feb;80(3):797–801. doi: 10.1073/pnas.80.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Minden M. D., Gusella J. F., Housman D. Chromosome-mediated transfer of the malignant phenotype by human acute myelogenous leukemic cells. Blood. 1984 Oct;64(4):842–846. [PubMed] [Google Scholar]
  19. 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]
  20. Nicolau C., Sene C. Liposome-mediated DNA transfer in eukaryotic cells. Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage. Biochim Biophys Acta. 1982 Oct 11;721(2):185–190. doi: 10.1016/0167-4889(82)90067-2. [DOI] [PubMed] [Google Scholar]
  21. Padmanabhan R., Corsico C. D., Howard T. H., Holter W., Fordis C. M., Willingham M., Howard B. H. Purification of transiently transfected cells by magnetic affinity cell sorting. Anal Biochem. 1988 May 1;170(2):341–348. doi: 10.1016/0003-2697(88)90640-9. [DOI] [PubMed] [Google Scholar]
  22. Padmanabhan R., Howard T. H., Howard B. H. Specific growth inhibitory sequences in genomic DNA from quiescent human embryo fibroblasts. Mol Cell Biol. 1987 May;7(5):1894–1899. doi: 10.1128/mcb.7.5.1894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Potter H., Weir L., Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161–7165. doi: 10.1073/pnas.81.22.7161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Satyabhama S., Epstein A. L. Short-term efficient expression of transfected DNA in human hematopoietic cells by electroporation: definition of parameters and use of chemical stimulators. DNA. 1988 Apr;7(3):203–209. doi: 10.1089/dna.1988.7.203. [DOI] [PubMed] [Google Scholar]
  25. Schaefer-Ridder M., Wang Y., Hofschneider P. H. Liposomes as gene carriers: efficient transformation of mouse L cells by thymidine kinase gene. Science. 1982 Jan 8;215(4529):166–168. doi: 10.1126/science.7053567. [DOI] [PubMed] [Google Scholar]
  26. Schaffner W. Direct transfer of cloned genes from bacteria to mammalian cells. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2163–2167. doi: 10.1073/pnas.77.4.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shen Y. M., Hirschhorn R. R., Mercer W. E., Surmacz E., Tsutsui Y., Soprano K. J., Baserga R. Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency. Mol Cell Biol. 1982 Sep;2(9):1145–1154. doi: 10.1128/mcb.2.9.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shimotohno K., Temin H. M. Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus. Cell. 1981 Oct;26(1 Pt 1):67–77. doi: 10.1016/0092-8674(81)90034-9. [DOI] [PubMed] [Google Scholar]
  29. Stow N. D., Wilkie N. M. An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J Gen Virol. 1976 Dec;33(3):447–458. doi: 10.1099/0022-1317-33-3-447. [DOI] [PubMed] [Google Scholar]
  30. Strain A. J., Wallace W. A., Wyllie A. H. Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells. Biochem J. 1985 Jan 15;225(2):529–533. doi: 10.1042/bj2250529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sutherland B. M., Bennett P. V. Transformation of human cells by DNA transfection. Cancer Res. 1984 Jul;44(7):2769–2772. [PubMed] [Google Scholar]
  32. Yamamoto F., Furusawa M., Furusawa I., Obinata M. The 'pricking' method. A new efficient technique for mechanically introducing foreign DNA into the nuclei of culture cells. Exp Cell Res. 1982 Nov;142(1):79–84. doi: 10.1016/0014-4827(82)90411-6. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES