Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Feb 15;24(4):543–548. doi: 10.1093/nar/24.4.543

Inducible site-directed recombination in mouse embryonic stem cells.

Y Zhang 1, C Riesterer 1, A M Ayrall 1, F Sablitzky 1, T D Littlewood 1, M Reth 1
PMCID: PMC145690  PMID: 8604292

Abstract

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly, the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins, tamoxifen can efficiently induce Cre-mediated recombination, thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen, recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein, the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals.

Full Text

The Full Text of this article is available as a PDF (91.5 KB).

Selected References

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

  1. Barinaga M. Knockout mice: round two. Science. 1994 Jul 1;265(5168):26–28. doi: 10.1126/science.8016653. [DOI] [PubMed] [Google Scholar]
  2. Burk O., Klempnauer K. H. Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein. EMBO J. 1991 Dec;10(12):3713–3719. doi: 10.1002/j.1460-2075.1991.tb04939.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bühler B., Köhler G., Nielsen P. J. A lacZ-based vector system for the rapid detection of V(D)J recombinase activity. J Immunol Methods. 1994 Oct 14;175(2):259–266. doi: 10.1016/0022-1759(94)90368-9. [DOI] [PubMed] [Google Scholar]
  4. Chambers C. A. TKO'ed: lox, stock and barrel. Bioessays. 1994 Dec;16(12):865–868. doi: 10.1002/bies.950161202. [DOI] [PubMed] [Google Scholar]
  5. Charlier C., Chariot A., Antoine N., Merville M. P., Gielen J., Castronovo V. Tamoxifen and its active metabolite inhibit growth of estrogen receptor-negative MDA-MB-435 cells. Biochem Pharmacol. 1995 Jan 31;49(3):351–358. doi: 10.1016/0006-2952(94)00492-5. [DOI] [PubMed] [Google Scholar]
  6. Cox M. M. The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4223–4227. doi: 10.1073/pnas.80.14.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Danielian P. S., White R., Hoare S. A., Fawell S. E., Parker M. G. Identification of residues in the estrogen receptor that confer differential sensitivity to estrogen and hydroxytamoxifen. Mol Endocrinol. 1993 Feb;7(2):232–240. doi: 10.1210/mend.7.2.8469236. [DOI] [PubMed] [Google Scholar]
  8. DeFriend D. J., Anderson E., Bell J., Wilks D. P., West C. M., Mansel R. E., Howell A. Effects of 4-hydroxytamoxifen and a novel pure antioestrogen (ICI 182780) on the clonogenic growth of human breast cancer cells in vitro. Br J Cancer. 1994 Aug;70(2):204–211. doi: 10.1038/bjc.1994.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DiSanto J. P., Müller W., Guy-Grand D., Fischer A., Rajewsky K. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):377–381. doi: 10.1073/pnas.92.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eilers M., Picard D., Yamamoto K. R., Bishop J. M. Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells. Nature. 1989 Jul 6;340(6228):66–68. doi: 10.1038/340066a0. [DOI] [PubMed] [Google Scholar]
  11. Gossen M., Bonin A. L., Bujard H. Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 1993 Dec;18(12):471–475. doi: 10.1016/0968-0004(93)90009-c. [DOI] [PubMed] [Google Scholar]
  12. Gossen M., Freundlieb S., Bender G., Müller G., Hillen W., Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science. 1995 Jun 23;268(5218):1766–1769. doi: 10.1126/science.7792603. [DOI] [PubMed] [Google Scholar]
  13. Gu H., Marth J. D., Orban P. C., Mossmann H., Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science. 1994 Jul 1;265(5168):103–106. doi: 10.1126/science.8016642. [DOI] [PubMed] [Google Scholar]
  14. Haran E. F., Maretzek A. F., Goldberg I., Horowitz A., Degani H. Tamoxifen enhances cell death in implanted MCF7 breast cancer by inhibiting endothelium growth. Cancer Res. 1994 Nov 1;54(21):5511–5514. [PubMed] [Google Scholar]
  15. Kilby N. J., Snaith M. R., Murray J. A. Site-specific recombinases: tools for genome engineering. Trends Genet. 1993 Dec;9(12):413–421. doi: 10.1016/0168-9525(93)90104-p. [DOI] [PubMed] [Google Scholar]
  16. Kühn R., Schwenk F., Aguet M., Rajewsky K. Inducible gene targeting in mice. Science. 1995 Sep 8;269(5229):1427–1429. doi: 10.1126/science.7660125. [DOI] [PubMed] [Google Scholar]
  17. Littlewood T. D., Hancock D. C., Danielian P. S., Parker M. G., Evan G. I. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 1995 May 25;23(10):1686–1690. doi: 10.1093/nar/23.10.1686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Logie C., Stewart A. F. Ligand-regulated site-specific recombination. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5940–5944. doi: 10.1073/pnas.92.13.5940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Metzger D., Clifford J., Chiba H., Chambon P. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6991–6995. doi: 10.1073/pnas.92.15.6991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Odell J. T., Hoopes J. L., Vermerris W. Seed-specific gene activation mediated by the Cre/lox site-specific recombination system. Plant Physiol. 1994 Oct;106(2):447–458. doi: 10.1104/pp.106.2.447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Picard D. Steroid-binding domains for regulating the functions of heterologous proteins in cis. Trends Cell Biol. 1993 Aug;3(8):278–280. doi: 10.1016/0962-8924(93)90057-8. [DOI] [PubMed] [Google Scholar]
  22. Pratt W. B. Interaction of hsp90 with steroid receptors: organizing some diverse observations and presenting the newest concepts. Mol Cell Endocrinol. 1990 Nov 12;74(1):C69–C76. doi: 10.1016/0303-7207(90)90198-h. [DOI] [PubMed] [Google Scholar]
  23. Randerath K., Moorthy B., Mabon N., Sriram P. Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite. Carcinogenesis. 1994 Oct;15(10):2087–2094. doi: 10.1093/carcin/15.10.2087. [DOI] [PubMed] [Google Scholar]
  24. Reth M., Petrac E., Wiese P., Lobel L., Alt F. W. Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J. 1987 Nov;6(11):3299–3305. doi: 10.1002/j.1460-2075.1987.tb02649.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Samuels M. L., Weber M. J., Bishop J. M., McMahon M. Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase. Mol Cell Biol. 1993 Oct;13(10):6241–6252. doi: 10.1128/mcb.13.10.6241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sauer B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2087–2096. doi: 10.1128/mcb.7.6.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sauer B., Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166–5170. doi: 10.1073/pnas.85.14.5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sauer B., Henderson N. Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biol. 1990 May;2(5):441–449. [PubMed] [Google Scholar]
  29. Schumacher M., Bastert G., Bojar H., Hübner K., Olschewski M., Sauerbrei W., Schmoor C., Beyerle C., Neumann R. L., Rauschecker H. F. Randomized 2 x 2 trial evaluating hormonal treatment and the duration of chemotherapy in node-positive breast cancer patients. German Breast Cancer Study Group. J Clin Oncol. 1994 Oct;12(10):2086–2093. doi: 10.1200/JCO.1994.12.10.2086. [DOI] [PubMed] [Google Scholar]
  30. Smith D. F., Toft D. O. Steroid receptors and their associated proteins. Mol Endocrinol. 1993 Jan;7(1):4–11. doi: 10.1210/mend.7.1.8446107. [DOI] [PubMed] [Google Scholar]
  31. Tsubata T., Reth M. The products of pre-B cell-specific genes (lambda 5 and VpreB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface. J Exp Med. 1990 Sep 1;172(3):973–976. doi: 10.1084/jem.172.3.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Williams M. L., Lennard M. S., Martin I. J., Tucker G. T. Interindividual variation in the isomerization of 4-hydroxytamoxifen by human liver microsomes: involvement of cytochromes P450. Carcinogenesis. 1994 Dec;15(12):2733–2738. doi: 10.1093/carcin/15.12.2733. [DOI] [PubMed] [Google Scholar]

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

RESOURCES