Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Aug;4(8):1644–1646. doi: 10.1128/mcb.4.8.1644

Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion.

A S Waldman, G Milman
PMCID: PMC368961  PMID: 6092926

Abstract

The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. We have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report we show that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of [3H]thymidine into cell nuclei as measured by autoradiography.

Full text

PDF
1644

Images in this article

1645Image
on p.1645

Selected References

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

  1. Diacumakos E. G., Holland S., Pecora P. A microsurgical methodology for human cells in vitro: evolution and applications. Proc Natl Acad Sci U S A. 1970 Apr;65(4):911–918. doi: 10.1073/pnas.65.4.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Furusawa M., Nishimura T., Yamaizumi M., Okada Y. Injection of foreign substances into single cells by cell fusion. Nature. 1974 May 31;249(456):449–450. doi: 10.1038/249449a0. [DOI] [PubMed] [Google Scholar]
  3. Kaltoft K., Celis J. E. Ghost-mediated transfer of human hypoxanthine-guanine phosphoribosyl transferase into deficient Chinese hamster ovary cells by means of polyethylene glycol-induced fusion. Exp Cell Res. 1978 Sep;115(2):423–428. doi: 10.1016/0014-4827(78)90299-9. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Liu S. W., Milman G. Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor. J Biol Chem. 1983 Jun 25;258(12):7469–7475. [PubMed] [Google Scholar]
  6. Loyter A., Zakai N., Kulka R. G. "Ultramicroinjection" of macromolecules or small particles into animal cells. A new technique based on virus-induced cell fusion. J Cell Biol. 1975 Aug;66(2):292–304. doi: 10.1083/jcb.66.2.292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Oi V. T., Morrison S. L., Herzenberg L. A., Berg P. Immunoglobulin gene expression in transformed lymphoid cells. Proc Natl Acad Sci U S A. 1983 Feb;80(3):825–829. doi: 10.1073/pnas.80.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Pagano R. E., Weinstein J. N. Interactions of liposomes with mammalian cells. Annu Rev Biophys Bioeng. 1978;7:435–468. doi: 10.1146/annurev.bb.07.060178.002251. [DOI] [PubMed] [Google Scholar]
  9. Rechsteiner M. C. Uptake of proteins by red blood cells. Exp Cell Res. 1975 Jul;93(2):487–492. doi: 10.1016/0014-4827(75)90478-4. [DOI] [PubMed] [Google Scholar]
  10. Sandri-Goldin R. M., Goldin A. L., Levine M., Glorioso J. C. High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion. Mol Cell Biol. 1981 Aug;1(8):743–752. doi: 10.1128/mcb.1.8.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Stacey D. W., Allfrey V. G., Hanafusa H. Microinjection analysis of envelope-glycoprotein messenger activities of avian leukosis viral RNAs. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1614–1618. doi: 10.1073/pnas.74.4.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Tjian R., Fey G., Graessmann A. Biological activity of purified simian virus 40 T antigen proteins. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1279–1283. doi: 10.1073/pnas.75.3.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Waldman A. S., Haeusslein E., Milman G. Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor. J Biol Chem. 1983 Oct 10;258(19):11571–11575. [PubMed] [Google Scholar]
  15. Weinstein J. N., Yoshikami S., Henkart P., Blumenthal R., Hagins W. A. Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker. Science. 1977 Feb 4;195(4277):489–492. doi: 10.1126/science.835007. [DOI] [PubMed] [Google Scholar]

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

RESOURCES