Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Oct;14(10):6936–6943. doi: 10.1128/mcb.14.10.6936

Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

P J Detloff 1, J Lewis 1, S W John 1, W R Shehee 1, R Langenbach 1, N Maeda 1, O Smithies 1
PMCID: PMC359224  PMID: 7935410

Abstract

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

Full text

PDF
6936

Images in this article

6939Image
on p.6939
6941Image
on p.6941

Selected References

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

  1. Askew G. R., Doetschman T., Lingrel J. B. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol Cell Biol. 1993 Jul;13(7):4115–4124. doi: 10.1128/mcb.13.7.4115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Behringer R. R., Ryan T. M., Reilly M. P., Asakura T., Palmiter R. D., Brinster R. L., Townes T. M. Synthesis of functional human hemoglobin in transgenic mice. Science. 1989 Sep 1;245(4921):971–973. doi: 10.1126/science.2772649. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Charnay P., Mellon P., Maniatis T. Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells. Mol Cell Biol. 1985 Jun;5(6):1498–1511. doi: 10.1128/mcb.5.6.1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cowie A., Myers R. M. DNA sequences involved in transcriptional regulation of the mouse beta-globin promoter in murine erythroleukemia cells. Mol Cell Biol. 1988 Aug;8(8):3122–3128. doi: 10.1128/mcb.8.8.3122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Kim H. S., Smithies O. Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 26;16(18):8887–8903. doi: 10.1093/nar/16.18.8887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Macleod K., Plumb M. Derepression of mouse beta-major-globin gene transcription during erythroid differentiation. Mol Cell Biol. 1991 Sep;11(9):4324–4332. doi: 10.1128/mcb.11.9.4324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Reid L. H., Gregg R. G., Smithies O., Koller B. H. Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4299–4303. doi: 10.1073/pnas.87.11.4299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ryan T. M., Townes T. M., Reilly M. P., Asakura T., Palmiter R. D., Brinster R. L., Behringer R. R. Human sickle hemoglobin in transgenic mice. Science. 1990 Feb 2;247(4942):566–568. doi: 10.1126/science.2154033. [DOI] [PubMed] [Google Scholar]
  18. Shehee W. R., Oliver P., Smithies O. Lethal thalassemia after insertional disruption of the mouse major adult beta-globin gene. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3177–3181. doi: 10.1073/pnas.90.8.3177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shesely E. G., Kim H. S., Shehee W. R., Papayannopoulou T., Smithies O., Popovich B. W. Correction of a human beta S-globin gene by gene targeting. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4294–4298. doi: 10.1073/pnas.88.10.4294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Skow L. C., Burkhart B. A., Johnson F. M., Popp R. A., Popp D. M., Goldberg S. Z., Anderson W. F., Barnett L. B., Lewis S. E. A mouse model for beta-thalassemia. Cell. 1983 Oct;34(3):1043–1052. doi: 10.1016/0092-8674(83)90562-7. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Stacey A., Schnieke A., McWhir J., Cooper J., Colman A., Melton D. W. Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice. Mol Cell Biol. 1994 Feb;14(2):1009–1016. doi: 10.1128/mcb.14.2.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Wu H., Liu X., Jaenisch R. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2819–2823. doi: 10.1073/pnas.91.7.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]

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

RESOURCES