Identification of genes differentially regulated by glucocorticoids and progestins using a Cre/loxP-mediated retroviral promoter-trapping strategy.
Glucocorticoids and progestins are two classes of steroid hormone with very distinct biological functions. However, the glucocorticoid receptor (GR) and the progesterone receptor (PR) share many structural and functional similarities. One way that glucocorticoids and progestins can exert different biological effects is through their different abilities to regulate the expression of certain target genes. A strategy employing a retroviral promoter-trap and Cre/loxP-mediated site-specific recombination has been developed to identify genes that are differentially regulated by glucocorticoids and progestins. A mouse fibroblast cell line (4F) stably expressing both GR and PR and containing a single copy of a multifunctional selection plasmid is generated. This line is transduced with a self-inactivating retroviral promoter-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Integration of the provirus places Cre expression under the control of a genomic flanking sequence. Activation of Cre expression from integration into active genes results in a permanent switch between the selectable marker genes that converts the cells from neomycin-resistant to hygromycin-resistant. Selection for hygromycin resistance after hormone treatment yields recombinants in which Cre sequences in the U3 region are expressed from hormone-inducible upstream cellular promoters. Because Cre-mediated recombination is a permanent event, the expression of the selectable marker genes is independent of ongoing Cre expression. Thus this system permits the identification of genes that are transiently or weakly induced by hormone.
Wan
Y
Y
Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Nordeen
S K
SK
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D005938:Glucocorticoids / Q000494:pharmacology*; D019426:Integrases / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D045167:Progesterone Congeners / Q000494:pharmacology*; D011397:Promegestone / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics; D011980:Receptors, Progesterone / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D005938:Glucocorticoids; D045167:Progesterone Congeners; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D014764:Viral Proteins; D003907:Dexamethasone; D011397:Promegestone; C045073:Cre recombinase; D019426:Integrases