Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Jan;12(1):360–367. doi: 10.1128/mcb.12.1.360

Gene replacement with one-sided homologous recombination.

N Berinstein 1, N Pennell 1, C A Ottaway 1, M J Shulman 1
PMCID: PMC364128  PMID: 1729610

Abstract

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

Full text

PDF
360

Images in this article

363Image
on p.363
364Image
on p.364
364Image
on p.364
364Image
on p.364
365Image
on p.365

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. Baker M. D. High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions. Mol Cell Biol. 1989 Dec;9(12):5500–5507. doi: 10.1128/mcb.9.12.5500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker M. D., Pennell N., Bosnoyan L., Shulman M. J. Homologous recombination can restore normal immunoglobulin production in a mutant hybridoma cell line. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6432–6436. doi: 10.1073/pnas.85.17.6432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baumann B., Potash M. J., Köhler G. Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse. EMBO J. 1985 Feb;4(2):351–359. doi: 10.1002/j.1460-2075.1985.tb03636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Doetschman T., Maeda N., Smithies O. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8583–8587. doi: 10.1073/pnas.85.22.8583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellis J., Bernstein A. Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology. Mol Cell Biol. 1989 Apr;9(4):1621–1627. doi: 10.1128/mcb.9.4.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fell H. P., Yarnold S., Hellström I., Hellström K. E., Folger K. R. Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8507–8511. doi: 10.1073/pnas.86.21.8507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hicks J. B., Hinnen A., Fink G. R. Properties of yeast transformation. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1305–1313. doi: 10.1101/sqb.1979.043.01.149. [DOI] [PubMed] [Google Scholar]
  10. Köhler G., Potash M. J., Lehrach H., Shulman M. J. Deletions in immunoglobulin mu chains. EMBO J. 1982;1(5):555–563. doi: 10.1002/j.1460-2075.1982.tb01208.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Marcu K. B., Banerji J., Penncavage N. A., Lang R., Arnheim N. 5' flanking region of immunoglobulin heavy chain constant region genes displays length heterogeneity in germlines of inbred mouse strains. Cell. 1980 Nov;22(1 Pt 1):187–196. doi: 10.1016/0092-8674(80)90167-1. [DOI] [PubMed] [Google Scholar]
  12. Murnane J. P., Yezzi M. J., Young B. R. Recombination events during integration of transfected DNA into normal human cells. Nucleic Acids Res. 1990 May 11;18(9):2733–2738. doi: 10.1093/nar/18.9.2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ochi A., Hawley R. G., Hawley T., Shulman M. J., Traunecker A., Köhler G., Hozumi N. Functional immunoglobulin M production after transfection of cloned immunoglobulin heavy and light chain genes into lymphoid cells. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6351–6355. doi: 10.1073/pnas.80.20.6351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Smith A. J., Kalogerakis B. Replacement recombinant events targeted at immunoglobulin heavy chain DNA sequences in mouse myeloma cells. J Mol Biol. 1990 Jun 5;213(3):415–435. doi: 10.1016/s0022-2836(05)80205-0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Subramani S. Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors. Mutat Res. 1989 Mar-May;220(2-3):221–234. doi: 10.1016/0165-1110(89)90026-2. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES