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. 1987 Jun;7(6):2286–2293. doi: 10.1128/mcb.7.6.2286

Poly-L-ornithine-mediated transformation of mammalian cells.

V C Bond, B Wold
PMCID: PMC365353  PMID: 3600662

Abstract

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

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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. Botchan M., Topp W., Sambrook J. The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell. 1976 Oct;9(2):269–287. doi: 10.1016/0092-8674(76)90118-5. [DOI] [PubMed] [Google Scholar]
  3. Capecchi M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. doi: 10.1016/0092-8674(80)90358-x. [DOI] [PubMed] [Google Scholar]
  4. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deisseroth A., Hendrick D. Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells. Cell. 1978 Sep;15(1):55–63. doi: 10.1016/0092-8674(78)90082-x. [DOI] [PubMed] [Google Scholar]
  6. Fraley R. T., Fornari C. S., Kaplan S. Entrapment of a bacterial plasmid in phospholipid vesicles: potential for gene transfer. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3348–3352. doi: 10.1073/pnas.76.7.3348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Hirth K. P., Edwards C. A., Firtel R. A. A DNA-mediated transformation system for Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7356–7360. doi: 10.1073/pnas.79.23.7356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoffman R. M., Margolis L. B., Bergelson L. D. Binding and entrapment of high molecular weight DNA by lecithin liposomes. FEBS Lett. 1978 Sep 15;93(2):365–368. doi: 10.1016/0014-5793(78)81141-7. [DOI] [PubMed] [Google Scholar]
  10. Jeffreys A. J., Flavell R. A. A physical map of the DNA regions flanking the rabbit beta-globin gene. Cell. 1977 Oct;12(2):429–439. doi: 10.1016/0092-8674(77)90119-2. [DOI] [PubMed] [Google Scholar]
  11. KIT S., DUBBS D. R., PIEKARSKI L. J., HSU T. C. DELETION OF THYMIDINE KINASE ACTIVITY FROM L CELLS RESISTANT TO BROMODEOXYURIDINE. Exp Cell Res. 1963 Aug;31:297–312. doi: 10.1016/0014-4827(63)90007-7. [DOI] [PubMed] [Google Scholar]
  12. Klebe R. J., Harriss J. V., Hanson D. P., Gauntt C. J. High-efficiency polyethylene glycol-mediated transformation of mammalian cells. Somat Cell Mol Genet. 1984 Sep;10(5):495–502. doi: 10.1007/BF01534854. [DOI] [PubMed] [Google Scholar]
  13. Koch G., Bishop J. M. The effect of polycations on the interaction of viral RNA with mammalian cells: studies on the infectivity of single- and double-stranded poliovirus RNA. Virology. 1968 May;35(1):9–17. doi: 10.1016/0042-6822(68)90300-0. [DOI] [PubMed] [Google Scholar]
  14. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kriegler M. P., Livingston D. M. Chemically facilitated microinjection of proteins into intact monolayers of tissue culture cells. Somatic Cell Genet. 1977 Nov;3(6):603–610. doi: 10.1007/BF01539068. [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. Lo C. W. Transformation by iontophoretic microinjection of DNA: multiple integrations without tandem insertions. Mol Cell Biol. 1983 Oct;3(10):1803–1814. doi: 10.1128/mcb.3.10.1803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lowy I., Pellicer A., Jackson J. F., Sim G. K., Silverstein S., Axel R. Isolation of transforming DNA: cloning the hamster aprt gene. Cell. 1980 Dec;22(3):817–823. doi: 10.1016/0092-8674(80)90558-9. [DOI] [PubMed] [Google Scholar]
  19. Lurquin P. F. Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts. Nucleic Acids Res. 1979 Aug 24;6(12):3773–3784. doi: 10.1093/nar/6.12.3773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Pagano J. S. Biologic activity of isolated viral nucleic acids. Prog Med Virol. 1970;12:1–48. [PubMed] [Google Scholar]
  23. Pellicer A., Robins D., Wold B., Sweet R., Jackson J., Lowy I., Roberts J. M., Sim G. K., Silverstein S., Axel R. Altering genotype and phenotype by DNA-mediated gene transfer. Science. 1980 Sep 19;209(4463):1414–1422. doi: 10.1126/science.7414320. [DOI] [PubMed] [Google Scholar]
  24. Pellicer A., Wigler M., Axel R., Silverstein S. The transfer and stable integration of the HSV thymidine kinase gene into mouse cells. Cell. 1978 May;14(1):133–141. doi: 10.1016/0092-8674(78)90308-2. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Rothstein S. J., Jorgensen R. A., Postle K., Reznikoff W. S. The inverted repeats of Tn5 are functionally different. Cell. 1980 Mar;19(3):795–805. doi: 10.1016/s0092-8674(80)80055-9. [DOI] [PubMed] [Google Scholar]
  27. SMULL C. E., LUDWIG E. H. Enhancement of the plaque-forming capacity of poliovirus ribonucleic acid with basic proteins. J Bacteriol. 1962 Nov;84:1035–1040. doi: 10.1128/jb.84.5.1035-1040.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Schlegel R. A., Rechsteiner M. C. Microinjection of thymidine kinase and bovine serum albumin into mammalian cells by fusion with red blood cells. Cell. 1975 Aug;5(4):371–379. doi: 10.1016/0092-8674(75)90056-2. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. Toneguzzo F., Hayday A. C., Keating A. Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells. Mol Cell Biol. 1986 Feb;6(2):703–706. doi: 10.1128/mcb.6.2.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tur-Kaspa R., Teicher L., Levine B. J., Skoultchi A. I., Shafritz D. A. Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. Mol Cell Biol. 1986 Feb;6(2):716–718. doi: 10.1128/mcb.6.2.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. White M. M., Mayne K. M., Lester H. A., Davidson N. Mouse-Torpedo hybrid acetylcholine receptors: functional homology does not equal sequence homology. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4852–4856. doi: 10.1073/pnas.82.14.4852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wiberg F. C., Sunnerhagen P., Kaltoft K., Zeuthen J., Bjursell G. Replication and expression in mammalian cells of transfected DNA; description of an improved erythrocyte ghost fusion technique. Nucleic Acids Res. 1983 Nov 11;11(21):7287–7302. doi: 10.1093/nar/11.21.7287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  38. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]

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