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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Dec 1;88(23):10416–10420. doi: 10.1073/pnas.88.23.10416

A dominant positive and negative selectable gene for use in mammalian cells.

F Schwartz 1, N Maeda 1, O Smithies 1, R Hickey 1, W Edelmann 1, A Skoultchi 1, R Kucherlapati 1
PMCID: PMC52939  PMID: 1720540

Abstract

We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusion protein that has TK and NEO protein functions. This and other fusion genes of a similar nature could serve as dominant positive and negative selectable markers in mammalian cells.

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Selected References

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  1. Colbere-Garapin F., Chousterman S., Horodniceanu F., Kourilsky P., Garapin A. C. Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3755–3759. doi: 10.1073/pnas.76.8.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Davies J., Smith D. I. Plasmid-determined resistance to antimicrobial agents. Annu Rev Microbiol. 1978;32:469–518. doi: 10.1146/annurev.mi.32.100178.002345. [DOI] [PubMed] [Google Scholar]
  3. Hasty P., Ramírez-Solis R., Krumlauf R., Bradley A. Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature. 1991 Mar 21;350(6315):243–246. doi: 10.1038/350243a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  6. Lowy D. R., Rands E., Scolnick E. M. Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J Virol. 1978 May;26(2):291–298. doi: 10.1128/jvi.26.2.291-298.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lupton S. D., Brunton L. L., Kalberg V. A., Overell R. W. Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene. Mol Cell Biol. 1991 Jun;11(6):3374–3378. doi: 10.1128/mcb.11.6.3374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mansour S. L., Thomas K. R., Capecchi M. R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. doi: 10.1038/336348a0. [DOI] [PubMed] [Google Scholar]
  9. McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  10. McKnight S. L. The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res. 1980 Dec 20;8(24):5949–5964. doi: 10.1093/nar/8.24.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  12. Reiss B., Sprengel R., Schaller H. Protein fusions with the kanamycin resistance gene from transposon Tn5. EMBO J. 1984 Dec 20;3(13):3317–3322. doi: 10.1002/j.1460-2075.1984.tb02297.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sedivy J. M., Sharp P. A. Positive genetic selection for gene disruption in mammalian cells by homologous recombination. Proc Natl Acad Sci U S A. 1989 Jan;86(1):227–231. doi: 10.1073/pnas.86.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Soeiro R., Darnell J. E. Competition hybridization by "pre-saturation" of HeLa cell DNA. J Mol Biol. 1969 Sep 28;44(3):551–562. doi: 10.1016/0022-2836(69)90379-9. [DOI] [PubMed] [Google Scholar]
  15. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  16. Thomas K. R., Capecchi M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503–512. doi: 10.1016/0092-8674(87)90646-5. [DOI] [PubMed] [Google Scholar]
  17. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Valancius V., Smithies O. Testing an "in-out" targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. Mol Cell Biol. 1991 Mar;11(3):1402–1408. doi: 10.1128/mcb.11.3.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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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