Quantitative Analysis of Gene Expression in Preimplantation Mouse Embryos Using Green Fluorescent Protein Reporter1

Abstract

We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluorescent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluorescence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available.

Knockdown of RLIM inhibits XIST expression and improves developmental competence of cloned male pig embryos

“…(2) The REX1 expression plasmid injected into cloned pig embryos at the one‐cell stage was degraded before ectopic activation of Xist expression, which was at the morula to blastocyst stages (5–7 days post‐activation) in cloned pig embryos (Yang et al, 2019; Zeng et al, 2016). This possibility was supported not only by our result showing that EGFP marker expression was not observed at the morula stage of cloned pig embryos injected with REX1 plasmid (Figure 5a), but also by some other studies showing that the expression of foreign genes from plasmid injected into embryos was maintained for less than 5 days (Hoelker et al, 2007; Medvedev et al, 2002; Sato et al, 2012). (3) The REX1 protein overexpressed in injected cloned pig embryos was rapidly degraded, as the half‐life of REX1 protein was shown to be less than 0.5 h in mouse cells with a normal RLIM gene (Gontan et al, 2012).…”

Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLCζ

“…When YFP-PLCζ was injected into eggs (pipette concentration up to 2 mg/ml) Ca 2+ oscillations were seen, but no YFP fluorescence was detected using whole cell or confocal fluorescent-imaging techniques (data not shown). The lack of signal with this chimeric protein is consistent with the fact that the lower threshold of detection for EGFP above autofluorescence in mouse eggs is approximately 0.6 µM (Medvedev et al, 2002), whereas the amount of PLCζ that is required to generate Ca 2+ oscillations is 0.3-6 nM. To investigate if PLCζ is indeed sequestered by the pronuclei, a more sensitive tagging method was required.…”