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- W2596804580 abstract "Lentiviral mediated transgenesis of trophectoderm in mouse blastocysts is a powerful means for exploring the roles of a candidate gene in placenta. When zona-denuded blastocysts are transduced with lentiviral vectors carrying a transgene, the tight junctions of the epithelial trophectoderm impede access to the inner cell mass and restrict transgenesis to trophoblast derivatives. However, the transgene may insert in genomic regions that eventually become heterochromatic, resulting in transgene silencing. In this study, we sought to determine the optimal dose of viral particles to overcome subsequent silencing, thereby providing uniform and consistent transgenesis of placental tissue. In an initial experiment, in vivo derived, zona-free, mouse blastocysts were co-incubated for 7 h with a lentivirus designed to express EGFP under the regulation of a constitutive EF1a promoter at three different viral concentrations (7, 70 and 350 ng/ml), with 40 blastocyst per group in three independent replicates. Uniform EGFP could be detected by fluorescence evaluation 24 h after viral transduction in the trophectoderm of all embryos exposed to 70 and 350 ng/ml virus but not after infection at 7 ng/ml. Quantitative RT-PCR confirmed expression of the transgene encoding EGFP expression in all experimental groups and was correlated with the viral concentration employed (ANOVA P<0.05; mean±SEM; 7 ng/ml 1±0.3 vs 70 ng/ml 4.59±1.1 vs 350 ng/ml 9.88±0.8). In a separate experiment, blastocysts were incubated with lentiviral vectors at the two higher concentrations and transferred to surrogate dams (30 blastocysts/group, 10 blastocysts/recipient). Recipients were sacrificed on D12 of pregnancy and conceptuses evaluated for EGFP expression. The number of viable fetuses did not differ between the two groups (24/30 in 70 ng/ml vs 20/30 in 350 ng/ml), but less than half of the placentas obtained from blastocysts incubated in 70 ng/ml (10/24) expressed EGFP, whereas all 20 placentas from the 350 ng/ml group showed green fluorescence. In conclusion, efficient transgene expression assessed at the blastocyst stage does not ensure later expression in placenta, and higher viral concentrations can overcome transgene silencing. Supported by a fellowship from the Lalor Foundation (PBA), and grants from the Preeclampsia Foundation (BT) and NIH HD067759 (RMR)." @default.
- W2596804580 created "2017-03-23" @default.
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- W2596804580 date "2012-08-01" @default.
- W2596804580 modified "2023-09-24" @default.
- W2596804580 title "Viral Titer Determines the Placental Expression of a Transgene Following Lentivirus-Mediated Transgenesis of Mouse Blastocysts." @default.
- W2596804580 doi "https://doi.org/10.1093/biolreprod/87.s1.412" @default.
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