Abstract
Currently, the efficient production of chimeric mice and their survival are still challenging. Recent researches have indicated that preimplantation embryo culture media and manipulation lead to abnormal methylation of histone in the H19/Igf2 promotor region and consequently alter their gene expression pattern. This investigation was designed to evaluate the relationship between the methylation state of histone H3 and H19/Igf2 expression in mice chimeric blastocysts. Mouse 129/Sv embryonic stem cells (mESCs) expressing the green fluorescent protein (mESCs-GFP) were injected into the perivitelline space of 2.5 days post-coitis (dpc) embryos (C57BL/6) using a micromanipulator. H3K4 and H3K9 methylation, and H19 and Igf2 expression was measured by immunocytochemistry and q-PCR, respectively, in blastocysts. Histone H3 trimethylation in H3K4 and H3K9 in chimeric blastocysts was significantly less and greater, respectively (p< 0.05), than in controls. H19 expression was significantly less (p< 0.05), while Igf2 expression was less, but not significantly so, in chimeric than in control blastocysts. Our results showed, that the alteration ofH3K4me3 and H3K9me3 methylation, change H19/Igf2 expression in chimeric blastocysts.
Highlights
Chimeras are animals composed of two or more genetically different cell lineages or recipient embryos from the same or different species [1]
Immunocytochemistry We used the Mouse 129/Sv embryonic stem cells (mESCs) carrying a GFP repoter to allow the chimeric blastocysts monitoring in the integration or exclusion during of the chimaera formation (Fig. 1.A)
The methylation of H3K4 and H3K9 in the blastocysts were visualized by immunocytochemistry (Fig. 1) and processed with using of Image J software
Summary
Chimeras are animals composed of two or more genetically different cell lineages or recipient embryos from the same or different species [1]. Previous studies showed that microinjection of ESCs into the blastocyst is an efficient approach to produce a good germ line-transmitted chimera [5,6,7] In this regard, application of laser technology to help introduce ESCs into the perivitelline embryonic space has its own advantage to produce ESC-derived F0 chimaeras [1]. Recent researches have indicated that preimplantation embryo culture media and manipulation lead to abnormal methylation of histone in the H19/Igf promotor region and alter their gene expression pattern. This investigation was designed to evaluate the relationship between the methylation state of histone H3 and H19/Igf expression in mice chimeric blastocysts. Conclusions: Our results showed, that the alteration ofH3K4me and H3K9me methylation, change H19/Igf expression in chimeric blastocysts
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