Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Jun 11;23(11):2058–2064. doi: 10.1093/nar/23.11.2058

Gene conversion during vector insertion in embryonic stem cells.

P Hasty 1, J Rivera-Pérez 1, A Bradley 1
PMCID: PMC306984  PMID: 7596837

Abstract

Recombination of an insertion vector into its chromosomal homologue is a conservative event in that both the chromosomal and the vector sequences are preserved. However, gene conversion may accompany homologous recombination of an insertion vector. To examine gene conversion in more detail we have determined the targeting frequencies and the structure of the recombinant alleles generated with a series of vectors which target the hprt gene in embryonic stem cells. We demonstrate that gene conversion of the introduced mutation does not significantly limit homologous recombination and that gene conversion occurs without a sequence specific bias for five different mutations. The frequency of the loss of a vector mutation and the gain of a chromosomal sequence is inversely proportional to the distance between the vector mutation and the double-strand break. The loss of a chromosomal sequence and the gain of a vector mutation occurs at a low frequency.

Full text

PDF
2060

Images in this article

Selected References

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

  1. Brenner D. A., Smigocki A. C., Camerini-Otero R. D. Double-strand gap repair results in homologous recombination in mouse L cells. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1762–1766. doi: 10.1073/pnas.83.6.1762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Deng C., Thomas K. R., Capecchi M. R. Location of crossovers during gene targeting with insertion and replacement vectors. Mol Cell Biol. 1993 Apr;13(4):2134–2140. doi: 10.1128/mcb.13.4.2134. [DOI] [PMC free article] [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. Hasty P., Rivera-Pérez J., Bradley A. The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol. 1991 Nov;11(11):5586–5591. doi: 10.1128/mcb.11.11.5586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hasty P., Rivera-Pérez J., Bradley A. The role and fate of DNA ends for homologous recombination in embryonic stem cells. Mol Cell Biol. 1992 Jun;12(6):2464–2474. doi: 10.1128/mcb.12.6.2464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hasty P., Rivera-Pérez J., Chang C., Bradley A. Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells. Mol Cell Biol. 1991 Sep;11(9):4509–4517. doi: 10.1128/mcb.11.9.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jasin M., Berg P. Homologous integration in mammalian cells without target gene selection. Genes Dev. 1988 Nov;2(11):1353–1363. doi: 10.1101/gad.2.11.1353. [DOI] [PubMed] [Google Scholar]
  9. Jessberger R., Berg P. Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system. Mol Cell Biol. 1991 Jan;11(1):445–457. doi: 10.1128/mcb.11.1.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McMahon A. P., Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell. 1990 Sep 21;62(6):1073–1085. doi: 10.1016/0092-8674(90)90385-r. [DOI] [PubMed] [Google Scholar]
  11. Melton D. W., Konecki D. S., Brennand J., Caskey C. T. Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2147–2151. doi: 10.1073/pnas.81.7.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Pennington S. L., Wilson J. H. Gene targeting in Chinese hamster ovary cells is conservative. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9498–9502. doi: 10.1073/pnas.88.21.9498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Resnick M. A. The repair of double-strand breaks in DNA; a model involving recombination. J Theor Biol. 1976 Jun;59(1):97–106. doi: 10.1016/s0022-5193(76)80025-2. [DOI] [PubMed] [Google Scholar]
  15. Rossignol J. L. Existence of homogeneous categories of mutants exhibiting various conversion patterns in gene 75 of Ascobolus immersus. Genetics. 1969 Dec;63(4):795–805. doi: 10.1093/genetics/63.4.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rossignol J. L., Paquette N. Disparity of gene conversion in frameshift mutants located in locus b2 of Ascobolus immersus. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2871–2875. doi: 10.1073/pnas.76.6.2871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shulman M. J., Nissen L., Collins C. Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol Cell Biol. 1990 Sep;10(9):4466–4472. doi: 10.1128/mcb.10.9.4466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Subramani S. Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination. Mol Cell Biol. 1986 Apr;6(4):1320–1325. doi: 10.1128/mcb.6.4.1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Umar A., Boyer J. C., Kunkel T. A. DNA loop repair by human cell extracts. Science. 1994 Nov 4;266(5186):814–816. doi: 10.1126/science.7973637. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Wilson J. H., Berget P. B., Pipas J. M. Somatic cells efficiently join unrelated DNA segments end-to-end. Mol Cell Biol. 1982 Oct;2(10):1258–1269. doi: 10.1128/mcb.2.10.1258. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES