Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Sep 25;20(18):4795–4801. doi: 10.1093/nar/20.18.4795

End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells.

Y Aratani 1, R Okazaki 1, H Koyama 1
PMCID: PMC334234  PMID: 1408793

Abstract

We have studied the mechanism of targeted recombination in mammalian cells using a hemizygous adenine phosphoribosyltransferase-deficient (APRT-) Chinese hamster ovary (CHO) cell mutant as a recipient. Three structurally different targeting vectors with a 5' or a 3', or both, end-deleted aprt sequence, in either a closed-circular or linear form, were transfected to the cells with a mutated aprt gene by electroporation. APRT-positive (APRT+) recombinant clones were selected and analyzed to study the gene correction events of the deletion mutation. Some half of 58 recombinant clones obtained resulted from corrections of the deleted chromosomal aprt gene by either gene replacement or gene insertion, a mechanism which is currently accepted for homologous recombination in mammalian cells. However, the chromosomal sequence in the remaining half of the recombinants remained uncorrected but their truncated end of the aprt gene in the incoming vectors was corrected by extending the end beyond the region of homology to the target locus; the corrected vector was then randomly integrated into the genome. This extension, termed end extension repair, was observed with all three vectors used and was as far as 4.6-kilobase (kb) or more long. It is evident that the novel repair reaction mediated by homologous recombination, in addition to gene replacement and gene insertion, is also involved in gene correction events in mammalian cells. We discuss the model which may account for this phenomenon.

Full text

PDF
4795

Images in this article

4797Image
on p.4797
4798Image
on p.4798
4798Image
on p.4798
4799Image
on p.4799
4799Image
on p.4799

Selected References

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

  1. Adair G. M., Nairn R. S., Wilson J. H., Seidman M. M., Brotherman K. A., MacKinnon C., Scheerer J. B. Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4574–4578. doi: 10.1073/pnas.86.12.4574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ayusawa D., Shimizu K., Koyama H., Takeishi K., Seno T. Unusual aspects of human thymidylate synthase in mouse cells introduced by DNA-mediated gene transfer. J Biol Chem. 1983 Jan 10;258(1):48–53. [PubMed] [Google Scholar]
  3. Bedale W. A., Inman R. B., Cox M. M. RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange. J Biol Chem. 1991 Apr 5;266(10):6499–6510. [PubMed] [Google Scholar]
  4. Belmaaza A., Wallenburg J. C., Brouillette S., Gusew N., Chartrand P. Genetic exchange between endogenous and exogenous LINE-1 repetitive elements in mouse cells. Nucleic Acids Res. 1990 Nov 11;18(21):6385–6391. doi: 10.1093/nar/18.21.6385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bollag R. J., Waldman A. S., Liskay R. M. Homologous recombination in mammalian cells. Annu Rev Genet. 1989;23:199–225. doi: 10.1146/annurev.ge.23.120189.001215. [DOI] [PubMed] [Google Scholar]
  6. Capecchi M. R. Altering the genome by homologous recombination. Science. 1989 Jun 16;244(4910):1288–1292. doi: 10.1126/science.2660260. [DOI] [PubMed] [Google Scholar]
  7. Doetschman T., Gregg R. G., Maeda N., Hooper M. L., Melton D. W., Thompson S., Smithies O. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987 Dec 10;330(6148):576–578. doi: 10.1038/330576a0. [DOI] [PubMed] [Google Scholar]
  8. Hama-Inaba H., Takahashi M., Kasai M., Shiomi T., Ito A., Hanaoka F., Sato K. Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension. Cell Struct Funct. 1987 Apr;12(2):173–180. doi: 10.1247/csf.12.173. [DOI] [PubMed] [Google Scholar]
  9. Joyner A. L., Skarnes W. C., Rossant J. Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells. Nature. 1989 Mar 9;338(6211):153–156. doi: 10.1038/338153a0. [DOI] [PubMed] [Google Scholar]
  10. Koyama H., Kodama H. Adenine phosphoribosyltransferase deficiency in cultured mouse mammary tumor FM3A cells resistant to 4-carbamoylimidazolium 5-olate. Cancer Res. 1982 Oct;42(10):4210–4214. [PubMed] [Google Scholar]
  11. Lin F. L., Sperle K., Sternberg N. Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Mol Cell Biol. 1984 Jun;4(6):1020–1034. doi: 10.1128/mcb.4.6.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Meselson M. S., Radding C. M. A general model for genetic recombination. Proc Natl Acad Sci U S A. 1975 Jan;72(1):358–361. doi: 10.1073/pnas.72.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nalbantoglu J., Hartley D., Phear G., Tear G., Meuth M. Spontaneous deletion formation at the aprt locus of hamster cells: the presence of short sequence homologies and dyad symmetries at deletion termini. EMBO J. 1986 Jun;5(6):1199–1204. doi: 10.1002/j.1460-2075.1986.tb04347.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Park J. H., Taylor M. W. Analysis of signals controlling expression of the Chinese hamster ovary aprt gene. Mol Cell Biol. 1988 Jun;8(6):2536–2544. doi: 10.1128/mcb.8.6.2536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Smithies O., Gregg R. G., Boggs S. S., Koralewski M. A., Kucherlapati R. S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985 Sep 19;317(6034):230–234. doi: 10.1038/317230a0. [DOI] [PubMed] [Google Scholar]
  17. Song K. Y., Schwartz F., Maeda N., Smithies O., Kucherlapati R. Accurate modification of a chromosomal plasmid by homologous recombination in human cells. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6820–6824. doi: 10.1073/pnas.84.19.6820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Soriano P., Montgomery C., Geske R., Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. doi: 10.1016/0092-8674(91)90499-o. [DOI] [PubMed] [Google Scholar]
  19. Stark G. R., Wahl G. M. Gene amplification. Annu Rev Biochem. 1984;53:447–491. doi: 10.1146/annurev.bi.53.070184.002311. [DOI] [PubMed] [Google Scholar]
  20. Szostak J. W., Orr-Weaver T. L., Rothstein R. J., Stahl F. W. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Thomas K. R., Folger K. R., Capecchi M. R. High frequency targeting of genes to specific sites in the mammalian genome. Cell. 1986 Feb 14;44(3):419–428. doi: 10.1016/0092-8674(86)90463-0. [DOI] [PubMed] [Google Scholar]
  23. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  24. Valancius V., Smithies O. Double-strand gap repair in a mammalian gene targeting reaction. Mol Cell Biol. 1991 Sep;11(9):4389–4397. doi: 10.1128/mcb.11.9.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Varshavsky A. On the possibility of metabolic control of replicon "misfiring": relationship to emergence of malignant phenotypes in mammalian cell lineages. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3673–3677. doi: 10.1073/pnas.78.6.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wong E. A., Capecchi M. R. Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays. Somat Cell Mol Genet. 1986 Jan;12(1):63–72. doi: 10.1007/BF01560728. [DOI] [PubMed] [Google Scholar]
  27. Zimmer A., Gruss P. Production of chimaeric mice containing embryonic stem (ES) cells carrying a homoeobox Hox 1.1 allele mutated by homologous recombination. Nature. 1989 Mar 9;338(6211):150–153. doi: 10.1038/338150a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES