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. 1986 Feb;6(2):716–718. doi: 10.1128/mcb.6.2.716

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

R Tur-Kaspa, L Teicher, B J Levine, A I Skoultchi, D A Shafritz
PMCID: PMC367564  PMID: 3466023

Abstract

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.

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Selected References

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

  1. Anderson W. F., Killos L., Sanders-Haigh L., Kretschmer P. J., Diacumakos E. G. Replication and expression of thymidine kinase and human globin genes microinjected into mouse fibroblasts. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5399–5403. doi: 10.1073/pnas.77.9.5399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  4. Enat R., Jefferson D. M., Ruiz-Opazo N., Gatmaitan Z., Leinwand L. A., Reid L. M. Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1411–1415. doi: 10.1073/pnas.81.5.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gordon J. W., Scangos G. A., Plotkin D. J., Barbosa J. A., Ruddle F. H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7380–7384. doi: 10.1073/pnas.77.12.7380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Isom H. C., Secott T., Georgoff I., Woodworth C., Mummaw J. Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci U S A. 1985 May;82(10):3252–3256. doi: 10.1073/pnas.82.10.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kucherlapati R., Skoultchi A. I. Introduction of purified genes into mammalian cells. CRC Crit Rev Biochem. 1984;16(4):349–379. doi: 10.3109/10409238409108719. [DOI] [PubMed] [Google Scholar]
  11. Laimins L. A., Khoury G., Gorman C., Howard B., Gruss P. Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6453–6457. doi: 10.1073/pnas.79.21.6453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Leffert H. L., Koch K. S., Moran T., Williams M. Liver cells. Methods Enzymol. 1979;58:536–544. doi: 10.1016/s0076-6879(79)58168-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Rassoulzadegan M., Binetruy B., Cuzin F. High frequency of gene transfer after fusion between bacteria and eukaryotic cells. Nature. 1982 Jan 21;295(5846):257–259. doi: 10.1038/295257a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Sodroski J., Rosen C., Wong-Staal F., Salahuddin S. Z., Popovic M., Arya S., Gallo R. C., Haseltine W. A. Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science. 1985 Jan 11;227(4683):171–173. doi: 10.1126/science.2981427. [DOI] [PubMed] [Google Scholar]
  20. Sompayrac L. M., Danna K. J. Efficient infection of monkey cells with DNA of simian virus 40. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7575–7578. doi: 10.1073/pnas.78.12.7575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. de Saint Vincent B. R., Delbrück S., Eckhart W., Meinkoth J., Vitto L., Wahl G. The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker. Cell. 1981 Dec;27(2 Pt 1):267–277. doi: 10.1016/0092-8674(81)90410-4. [DOI] [PubMed] [Google Scholar]

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