Background: Our laboratory utilizes murine models to study the effect of abnormalities in blood coagulation on placental function and pregnancy outcome. For these studies, a genetic tool was needed to knock out the expression of specific genes from the placenta at different stages of murine pregnancy. A Cre-Lox system was tested where male ERT2Cre mice were crossed to ROSA mT/mG reporter mice. ERT2Cre mice carry a transgene expressing the Cre recombinase fused to the ligand binding domain of estrogen receptor T2. The fusion protein remains in the cytoplasm until tamoxifen, an estrogen, binds to ERT2 and allows the complex to enter the nucleus. The reporter ROSA mT/mG mice carry a two-color sequence placed behind a strong constitutive promoter and targeted to the endogenous GTROSA locus. This reporter sequence expresses a membrane-targeted red fluorescent protein (mT) in all tissues due to a tdTomato cassette flanked by loxP sites. A membrane-targeted EGFP (mG) cassette is located downstream of the mT cassette and is expressed whenever loxP flanked mT cassette is excised. Thus, mTmG mice serve to report Cre-mediated excision in tissues by switching expression from red to green fluorescence. The goal of this experiment was to determine if treatment of the pregnant female with tamoxifen results in Cre activity in the mouse placenta and embryo, and to assess the penetrance of Cre activity. Methods: ROSA mTmGtg/tg female mice were mated to ERT2Cretg/tg male mice. All embryos and placentae from this genetic cross are expected to carry both, ERT2Cre and ROSA mTmG, sequences. Pregnant females were injected with 75 mg/kg tamoxifen at 8.5 days post coitum (dpc) and dissected at 12.5, 15.5, and 18.5 dpc. Embryonic and placental phenotypes were observed and analyzed by histology for fluorescence. Results: Embryos/placentae at 12.5 dpc displayed near-complete penetrance of tamoxifen-induced Cre-mediated excision, resulting in predominantly green fluorescent placentae and embryos. In contrast, pregnancies at 15.5 and 18.5 dpc showed mosaic green and red fluorescence. A large proportion of embryos showed deformities or underdevelopment. Conclusion: Our data shows that upon treatment of the pregnant female, tamoxifen crosses the placental barrier and allows effective Cre-mediated excision in placental and embryonic tissues. More pregnancies are being analyzed to examine dose- and stage- dependent effects of tamoxifen on Cre-mediated excision and embryonic development.

Measuring tamoxifen-inducible Cre expression in placental and embryonic tissues following treatment of pregnant female mice

255th National Meeting (2018)

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