Determination of Allelic Expression of H19 in Pre- and Peri-Implantation Mouse Embryos1
Determination of Allelic Expression of H19 in Pre- and Peri-Implantation Mouse Embryos1
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DOI：
10.1095/biolreprod.112.105882
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发表时间：
2012-08
期刊：
影响因子：
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通讯作者：
V. Negrón-Pérez;Franklin D. Echevarria;S. Huffman;R. M. Rivera
中科院分区：
文献类型：
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作者：
V. Negrón-Pérez;Franklin D. Echevarria;S. Huffman;R. M. Rivera
摘要H19是一种母源性表达的非编码RNA，具有抑癌活性。在小鼠着床前发育过程中，H19主要在滋养外胚层细胞中表达。本项目的目的是确定H19等位基因在植入前和围植入期小鼠胚胎中的表达。我们进一步感兴趣的是，确定是否在胚泡发育过程中发生的超数排卵和/或文化的结果印记H19表达的损失。我们的最后一个目标是确定是否差异H19等位基因表达之间发生的内细胞团（ICM）的一半和初级滋养层巨细胞（PTGC）的一半胚胎。在自然排卵或超数排卵后84、96和108 h从子宫收集C57 BL/6 J（Cast-7）xC 57 BL/6 J F1胚胎。在2-细胞期从输卵管收获体外培养的F1胚胎，并在补充有氨基酸的KSOM + aa或Whitten培养基中培养，并在上述时间收集。通过qRT-PCR随后荧光共振电子转移或RT-PCR随后限制性片段长度多态性和聚丙烯酰胺凝胶电泳（RFLP-PAGE）确定单个胚胎中等位基因特异性H19表达。将围着床期胚胎显微切割成两个切片，一个含有ICM，另一个含有PTGC。使用Dek、Pou 5 f1、Itga 7、H19和Igf 2的TaqMan探针来确定每个切片中的基因表达富集。通过RFLP-PAGE测定胚胎切片中等位基因特异性H19的表达。我们发现，随着胚胎在植入前发育中的进展，它们开始以双等位基因的方式表达H19，并且在培养的和体内发育的胚胎中观察到这种现象。如通过qRT-PCR测定的，当与含ICM的一半胚胎相比时，含PTGC的一半胚胎具有更高的H19表达。总之，H19印迹的损失发生在含PTGC的部分围着床期小鼠胚胎。我们推测这是小鼠植入时生理事件的一部分。
ABSTRACT 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.