Targeting of GFP-Cre to the Mouse Cyp11a1 Locus Both Drives Cre Recombinase Expression in Steroidogenic Cells and Permits Generation of Cyp11a1 Knock Out Mice

Abstract

To permit conditional gene targeting of floxed alleles in steroidogenic cell-types we have generated a transgenic mouse line that expresses Cre Recombinase under the regulation of the endogenous Cytochrome P450 side chain cleavage enzyme (Cyp11a1) promoter. Mice Carrying the Cyp11a1-GC (GFP-Cre) allele express Cre Recombinase in fetal adrenal and testis, and adrenal cortex, testicular Leydig cells (and a small proportion of Sertoli cells), theca cells of the ovary, and the hindbrain in postnatal life. Circulating testosterone concentration is unchanged in Cyp11+/GC males, suggesting steroidogenesis is unaffected by loss of one allele of Cyp11a1, mice are grossly normal, and Cre Recombinase functions to recombine floxed alleles of both a YFP reporter gene and the Androgen Receptor (AR) in steroidogenic cells of the testis, ovary, adrenal and hindbrain. Additionally, when bred to homozygosity (Cyp11a1GC/GC), knock-in of GFP-Cre to the endogenous Cyp11a1 locus results in a novel mouse model lacking endogenous Cyp11a1 (P450-SCC) function. This unique dual-purpose model has utility both for those wishing to conditionally target genes within steroidogenic cell types and for studies requiring mice lacking endogenous steroid hormone production.

Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

“…The fetal Leydig cell population was distinguished by hi TR expression (TR hi/GFP hi), whereas the other GFP-hi population expressed little TR, which allowed discrimination of another cell population that included Sertoli cells (TR lo/GFP hi). Sertoli cells are the first cells to differentiate within fetal testes (by E11.5), and a recent report that evaluated Cyp11a1-Cre activity documented GFP within Sertoli cells in adult testes [33], supporting early Cre activity. Statistical analyses supported the distinction between the two GFP-expressing populations.…”

“…A genetic approach was used to identify the fetal Leydig cell population within developing testes. Cyp11a1-Cre mice [33] were bred to mT/mG reporter mice [32] to cause excision of membrane-tagged tandem-dimer TR (mT) followed by expression of membrane-tagged enhanced GFP (mG) within steroidogenic cells. Endogenous fluorescence was visualized in whole gonads and adrenal cells under a dissecting microscope, which exhibited diffuse mT with punctate mGemitting cells (Fig.…”

“…Cyp11a1-Cre mice were made by targeting a GFP-Cre vector into the endogenous Cyp11a1 locus [33]. Cytoplasmic GFP expression was expected but undetectable after visualization under a fluorescence microscope or via IHC at any age in these mice [33].…”

“…Cytoplasmic GFP expression was expected but undetectable after visualization under a fluorescence microscope or via IHC at any age in these mice [33]. As a precaution, however, we chose to breed Cyp11a1-GFP-Cre (herein referred to as Cyp11a1-Cre) mice to the mT/mG reporter line so that the membrane-tagged GFP (mG) would be expressed along with cytoplasmic GFP in fetal Leydig cells of Cyp11a1-Cre;mT/mG embryos.…”

“…Our goal was to capitalize on microtechnology to discover molecular targets of fetal testis steroidogenesis so that we might prevent and treat disorders of masculinization that impact both fetal and adult male health. ; stock 007576; Jackson Laboratory, Bar Harbor, MD) [32] were bred to heterozygous Cyp11a1-Cre mice (B6;129P-Cyp11a1 tm1 [GFP/cre] Pzg /J; stock 010988; Jackson Laboratory) [33]. The presence of a vaginal plug the next morning was designated gestation or embryonic Day 0.5 (E0.5).…”