Fiche publication
Date publication
février 2001
Journal
Proceedings of the National Academy of Sciences of the United States of America
Auteurs
Membres identifiés du Cancéropôle Est :
Pr CHAMBON Pierre
,
Dr METZGER Daniel
Tous les auteurs :
Vallier L, Mancip J, Markossian S, Lukaszewicz A, Dehay C, Metzger D, Chambon P, Samarut J, Savatier P
Lien Pubmed
Résumé
We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the beta-galactosidase/neo(R) fusion protein and the Cre-ER(T2) (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygro(R) protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4'hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4'hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.
Mots clés
Alkaline Phosphatase, genetics, Animals, Base Sequence, Cell Differentiation, Cells, Cultured, Chimera, DNA Primers, Embryo, Mammalian, cytology, Gene Expression, drug effects, Genes, Reporter, Mice, Mice, Inbred BALB C, Stem Cells, metabolism, Tamoxifen, pharmacology
Référence
Proc. Natl. Acad. Sci. U.S.A.. 2001 Feb;98(5):2467-72
