Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.
We have developed a strategy to generate mutant genes in mammalian cells in a conditional manner by employing a fusion protein, Cre-ER, consisting of the loxP site-specific Cre recombinase linked to the ligand-binding domain of the human estrogen receptor. We have established homozygous retinoid X receptor alpha-negative (RXR alpha-/-) F9 embryonal carcinoma cells constitutively expressing Cre-ER and have shown that estradiol or the estrogen agonist/antagonist 4-hydroxytamoxifen efficiently induced the recombinase activity, whereas no activity was detected in the absence of ligand or in the presence of the antiestrogen ICI 164,384. Furthermore, using a targeting vector containing a selection marker flanked by loxP sites, we have inactivated one retinoic acid receptor alpha allele in such a line, demonstrating that the presence of the recombinase does not inhibit homologous recombination. Combining this conditional site-specific recombination system with tissue-specific expression of Cre-ER may allow modification of the mammalian genome in vivo in a spatiotemporally regulated manner.
Metzger
D
D
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université Louis Pasteur, Collège de France, Illkirch, C.U. de Strasbourg.
Clifford
J
J
Chiba
H
H
Chambon
P
P
D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004790:Enzyme Induction; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D018390:Gene Targeting; D005822:Genetic Vectors; D019426:Integrases; D016297:Mutagenesis, Site-Directed; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
D004965:Estrogen Antagonists; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; C051781:ICI 164384; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases