Transgenic production of the mouse in vitro- and in vivo-derived embryos: Effect on the methylation pattern of OCT4 promoter, expression levels of Dnmts, and Oct4

Abstract

We examined the methylation pattern of OCT4 promotor and expression levels of Dnmts and Oct4 genes in the transgenic mouse embryos obtained by in vivo and in vitro experiments. A gene construct consisting of selected parts of the region upstream from the human OCT4 promoter and enhanced green fluorescent protein as a reporter (OCT4-EGFP) was used for pronuclear microinjection into in vitro and in vivo-derived embryos. The rate of fertilization, cleavage and developmental competence of embryos was evaluated. Expression levels of targeted genes were investigated. DNA was extracted from embryos and treated using a bisulfite kit, and OCT4 methylation detection was done by PCR in both groups. After microinjection, GFP fluorescence was visualized in developing embryos. We observed a significant decrease in cleavage and blastulation rate in the IVM group compared with the in vivo group. Results showed higher gene expression for the selected genes in the in vivo embryos compare to the IVM. The band intensity of the PCR product loaded was different in both groups, which shows that the level of methylation is unlike in IVM and in vivo groups. DNA methylation during development plays an important role in embryonic development for the production of high-quality transgenic embryos.