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. 1994 Dec;14(12):8385–8390. doi: 10.1128/mcb.14.12.8385

Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.

P Hasty 1, M Crist 1, M Grompe 1, A Bradley 1
PMCID: PMC359377  PMID: 7969173

Abstract

We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.

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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. Anderson R. A., Eliason S. L. Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing. Mol Cell Biol. 1986 Sep;6(9):3246–3252. doi: 10.1128/mcb.6.9.3246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berinstein N., Pennell N., Ottaway C. A., Shulman M. J. Gene replacement with one-sided homologous recombination. Mol Cell Biol. 1992 Jan;12(1):360–367. doi: 10.1128/mcb.12.1.360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bollag R. J., Liskay R. M. Direct-repeat analysis of chromatid interactions during intrachromosomal recombination in mouse cells. Mol Cell Biol. 1991 Oct;11(10):4839–4845. doi: 10.1128/mcb.11.10.4839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Brenner D. A., Smigocki A. C., Camerini-Otero R. D. Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells. Mol Cell Biol. 1985 Apr;5(4):684–691. doi: 10.1128/mcb.5.4.684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brickell P. M. The p60c-src family of protein-tyrosine kinases: structure, regulation, and function. Crit Rev Oncog. 1992;3(4):401–446. [PubMed] [Google Scholar]
  8. Chakrabarti S., Seidman M. M. Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative. Mol Cell Biol. 1986 Jul;6(7):2520–2526. doi: 10.1128/mcb.6.7.2520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deng C., Capecchi M. R. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol. 1992 Aug;12(8):3365–3371. doi: 10.1128/mcb.12.8.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grompe M., al-Dhalimy M. Nucleotide sequence of a cDNA encoding murine fumarylacetoacetate hydrolase. Biochem Med Metab Biol. 1992 Aug;48(1):26–31. doi: 10.1016/0885-4505(92)90044-y. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Kang Y., Shulman M. J. Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells. Somat Cell Mol Genet. 1991 Nov;17(6):525–536. doi: 10.1007/BF01233617. [DOI] [PubMed] [Google Scholar]
  16. Koller B. H., Kim H. S., Latour A. M., Brigman K., Boucher R. C., Jr, Scambler P., Wainwright B., Smithies O. Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10730–10734. doi: 10.1073/pnas.88.23.10730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kucherlapati R. S., Eves E. M., Song K. Y., Morse B. S., Smithies O. Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA. Proc Natl Acad Sci U S A. 1984 May;81(10):3153–3157. doi: 10.1073/pnas.81.10.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lin F. L., Sperle K., Sternberg N. Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products. Mol Cell Biol. 1990 Jan;10(1):103–112. doi: 10.1128/mcb.10.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lin F. L., Sperle K., Sternberg N. Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells. Mol Cell Biol. 1990 Jan;10(1):113–119. doi: 10.1128/mcb.10.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maryon E., Carroll D. Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination. Mol Cell Biol. 1991 Jun;11(6):3278–3287. doi: 10.1128/mcb.11.6.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maryon E., Carroll D. Involvement of single-stranded tails in homologous recombination of DNA injected into Xenopus laevis oocyte nuclei. Mol Cell Biol. 1991 Jun;11(6):3268–3277. doi: 10.1128/mcb.11.6.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Ramírez-Solis R., Rivera-Pérez J., Wallace J. D., Wims M., Zheng H., Bradley A. Genomic DNA microextraction: a method to screen numerous samples. Anal Biochem. 1992 Mar;201(2):331–335. doi: 10.1016/0003-2697(92)90347-a. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Schultes N. P., Szostak J. W. A poly(dA.dT) tract is a component of the recombination initiation site at the ARG4 locus in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):322–328. doi: 10.1128/mcb.11.1.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seidman M. M. Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative. Mol Cell Biol. 1987 Oct;7(10):3561–3565. doi: 10.1128/mcb.7.10.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Strathern J. N., Klar A. J., Hicks J. B., Abraham J. A., Ivy J. M., Nasmyth K. A., McGill C. Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus. Cell. 1982 Nov;31(1):183–192. doi: 10.1016/0092-8674(82)90418-4. [DOI] [PubMed] [Google Scholar]
  33. Sun H., Treco D., Szostak J. W. Extensive 3'-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site. Cell. 1991 Mar 22;64(6):1155–1161. doi: 10.1016/0092-8674(91)90270-9. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Thomas K. R., Deng C., Capecchi M. R. High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol Cell Biol. 1992 Jul;12(7):2919–2923. doi: 10.1128/mcb.12.7.2919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Wake C. T., Vernaleone F., Wilson J. H. Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells. Mol Cell Biol. 1985 Aug;5(8):2080–2089. doi: 10.1128/mcb.5.8.2080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang H., Hasty P., Bradley A. Targeting frequency for deletion vectors in embryonic stem cells. Mol Cell Biol. 1994 Apr;14(4):2404–2410. doi: 10.1128/mcb.14.4.2404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. te Riele H., Maandag E. R., Berns A. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5128–5132. doi: 10.1073/pnas.89.11.5128. [DOI] [PMC free article] [PubMed] [Google Scholar]

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