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13 March 2013 Determination of Allelic Expression of H19 in Pre- and Peri-Implantation Mouse Embryos
Verónica M. Negrón-Pérez, Franklin D. Echevarría, Sarah R. Huffman, Rocío Melissa Rivera
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H19 is a maternally expressed, imprinted, noncoding RNA with tumor-suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. The purpose of this project was to determine allelic expression of H19 in pre- and peri-implantation mouse embryos. We were further interested in determining if loss of imprinted H19 expression during blastocyst development occurred as a result of superovulation and/or culture. Our last goal was to ascertain if differential H19 allelic expression occurred between the inner cell mass (ICM)-containing half and the primary trophoblast giant cell (PTGC)-containing half of the embryo. C57BL/6J(Cast-7)xC57BL/6J F1 embryos were collected from the uterus at 84, 96, and 108 h following natural ovulation or superovulation. In vitro-cultured F1 embryos were harvested from the oviduct at the 2-cell stage and cultured in KSOM aa supplemented with amino acids or Whitten media and collected at the above-mentioned times. Allele-specific H19 expression in single embryos was determined by qRT-PCR followed by fluorescence resonance electron transfer or RT-PCR followed by restriction fragment length polymorphism and polyacrylamide gel electrophoresis (RFLP-PAGE). Peri-implantation embryos were microdissected into two sections, one containing the ICM and the other containing the PTGC. TaqMan probes for Dek, Pou5f1, Itga7, H19, and Igf2 were used to ascertain gene expression enrichment in each section. Allele-specific H19 expression in embryo sections was determined by RFLP-PAGE. We found that as embryos advance through preimplantation development they start expressing H19 in a biallelic manner and this phenomenon was observed in the cultured and the in vivo-developed embryos. The PTGC-containing half of the embryo had greater expression of H19 when compared to the ICM-containing half of the embryo, as determined by qRT-PCR. In conclusion, loss of imprinting of H19 occurs in the PTGC-containing section of peri-implantation mouse embryos. We speculate that this is part of a physiologic event at the time of implantation in the mouse.

Verónica M. Negrón-Pérez, Franklin D. Echevarría, Sarah R. Huffman, and Rocío Melissa Rivera "Determination of Allelic Expression of H19 in Pre- and Peri-Implantation Mouse Embryos," Biology of Reproduction 88(4), (13 March 2013).
Received: 29 October 2012; Accepted: 1 March 2013; Published: 13 March 2013
DNA methylation
embryo culture
primary trophoblast giant cells
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