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. 1992 Jun 1;89(11):5128–5132. doi: 10.1073/pnas.89.11.5128

Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs.

H te Riele 1, E R Maandag 1, A Berns 1
PMCID: PMC49242  PMID: 1594621

Abstract

A vast amount of data suggests that homologous recombination in mammalian cells is relatively rare as compared to random integration, imposing the need for sophisticated selection protocols to enrich for cells in which homologous recombination has occurred. We here show that one of the key factors in efficient homologous recombination is the use of isogenic DNA to prepare the targeting vectors. Homologous recombination at the retinoblastoma susceptibility gene (Rb) in embryonic stem cells derived from mouse strain 129 was 20-fold more efficient with a 129-derived targeting construct than with a BALB/c-derived construct. The two constructs were identical, except for a number of base sequence divergences between 129 and BALB/c DNA, including base-pair substitutions, small deletions/insertions, and a polymorphic CA repeat. Transfection with an isogenic DNA construct, containing 17 kilobases of homology, yielded a targeting frequency of 78% (of a total of 20,000 drug-resistant colonies), without the use of an enrichment protocol for homologous recombination. This result indicates that, also in mammalian cells, homologous recombination rather than random integration can be the predominant event.

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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. Bernards R., Shackleford G. M., Schackleford G. M., Gerber M. R., Horowitz J. M., Friend S. H., Schartl M., Bogenmann E., Rapaport J. M., McGee T. Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6474–6478. doi: 10.1073/pnas.86.17.6474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishop C. E., Boursot P., Baron B., Bonhomme F., Hatat D. Most classical Mus musculus domesticus laboratory mouse strains carry a Mus musculus musculus Y chromosome. Nature. 1985 May 2;315(6014):70–72. doi: 10.1038/315070a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Brooks P., Dohet C., Almouzni G., Méchali M., Radman M. Mismatch repair involving localized DNA synthesis in extracts of Xenopus eggs. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4425–4429. doi: 10.1073/pnas.86.12.4425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Camerini-Otero R. D., Kucherlapati R. Right on target. Applications of Homologous Recombination to Human Disease sponsored by the National Institutes of Health (NIGMS, NIDDK, and NICHD) Bethesda, MD, USA, November 6-8, 1989. New Biol. 1990 Apr;2(4):337–341. [PubMed] [Google Scholar]
  7. 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]
  8. Capecchi M. R. The new mouse genetics: altering the genome by gene targeting. Trends Genet. 1989 Mar;5(3):70–76. doi: 10.1016/0168-9525(89)90029-2. [DOI] [PubMed] [Google Scholar]
  9. Charron J., Malynn B. A., Robertson E. J., Goff S. P., Alt F. W. High-frequency disruption of the N-myc gene in embryonic stem and pre-B cell lines by homologous recombination. Mol Cell Biol. 1990 Apr;10(4):1799–1804. doi: 10.1128/mcb.10.4.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Claverys J. P., Lacks S. A. Heteroduplex deoxyribonucleic acid base mismatch repair in bacteria. Microbiol Rev. 1986 Jun;50(2):133–165. doi: 10.1128/mr.50.2.133-165.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holmes J., Jr, Clark S., Modrich P. Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5837–5841. doi: 10.1073/pnas.87.15.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hooper M., Hardy K., Handyside A., Hunter S., Monk M. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature. 1987 Mar 19;326(6110):292–295. doi: 10.1038/326292a0. [DOI] [PubMed] [Google Scholar]
  13. Johnson R. S., Sheng M., Greenberg M. E., Kolodner R. D., Papaioannou V. E., Spiegelman B. M. Targeting of nonexpressed genes in embryonic stem cells via homologous recombination. Science. 1989 Sep 15;245(4923):1234–1236. doi: 10.1126/science.2506639. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Koller B. H., Hagemann L. J., Doetschman T., Hagaman J. R., Huang S., Williams P. J., First N. L., Maeda N., Smithies O. Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8927–8931. doi: 10.1073/pnas.86.22.8927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koller B. H., Smithies O. Inactivating the beta 2-microglobulin locus in mouse embryonic stem cells by homologous recombination. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8932–8935. doi: 10.1073/pnas.86.22.8932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kramer B., Kramer W., Williamson M. S., Fogel S. Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes. Mol Cell Biol. 1989 Oct;9(10):4432–4440. doi: 10.1128/mcb.9.10.4432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lee W. H., Bookstein R., Hong F., Young L. J., Shew J. Y., Lee E. Y. Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science. 1987 Mar 13;235(4794):1394–1399. doi: 10.1126/science.3823889. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. McGee T. L., Yandell D. W., Dryja T. P. Structure and partial genomic sequence of the human retinoblastoma susceptibility gene. Gene. 1989 Aug 1;80(1):119–128. doi: 10.1016/0378-1119(89)90256-4. [DOI] [PubMed] [Google Scholar]
  21. Rayssiguier C., Thaler D. S., Radman M. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature. 1989 Nov 23;342(6248):396–401. doi: 10.1038/342396a0. [DOI] [PubMed] [Google Scholar]
  22. Reid L. H., Shesely E. G., Kim H. S., Smithies O. Cotransformation and gene targeting in mouse embryonic stem cells. Mol Cell Biol. 1991 May;11(5):2769–2777. doi: 10.1128/mcb.11.5.2769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rubnitz J., Subramani S. The minimum amount of homology required for homologous recombination in mammalian cells. Mol Cell Biol. 1984 Nov;4(11):2253–2258. doi: 10.1128/mcb.4.11.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Staats J. Standardized nomenclature for inbred strains of mice: seventh listing for the International Committee on Standardized Genetic Nomenclature for Mice. Cancer Res. 1980 Jul;40(7):2083–2128. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Thompson S., Clarke A. R., Pow A. M., Hooper M. L., Melton D. W. Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell. 1989 Jan 27;56(2):313–321. doi: 10.1016/0092-8674(89)90905-7. [DOI] [PubMed] [Google Scholar]
  31. Waldman A. S., Liskay R. M. Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology. Mol Cell Biol. 1988 Dec;8(12):5350–5357. doi: 10.1128/mcb.8.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yenofsky R. L., Fine M., Pellow J. W. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci U S A. 1990 May;87(9):3435–3439. doi: 10.1073/pnas.87.9.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zijlstra M., Li E., Sajjadi F., Subramani S., Jaenisch R. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells. Nature. 1989 Nov 23;342(6248):435–438. doi: 10.1038/342435a0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. te Riele H., Maandag E. R., Clarke A., Hooper M., Berns A. Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells. Nature. 1990 Dec 13;348(6302):649–651. doi: 10.1038/348649a0. [DOI] [PubMed] [Google Scholar]
  36. van der Lugt N., Maandag E. R., te Riele H., Laird P. W., Berns A. A pgk::hprt fusion as a selectable marker for targeting of genes in mouse embryonic stem cells: disruption of the T-cell receptor delta-chain-encoding gene. Gene. 1991 Sep 15;105(2):263–267. doi: 10.1016/0378-1119(91)90161-4. [DOI] [PubMed] [Google Scholar]

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