Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage.
Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage.
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DOI：
10.1530/rep-07-0211
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发表时间：
2008
期刊：
影响因子：
3.8
通讯作者：
A. Thurston;Jane E. Taylor;J. Gardner;K. Sinclair;L. Young
中科院分区：
文献类型：
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作者：
A. Thurston;Jane E. Taylor;J. Gardner;K. Sinclair;L. Young
一系列哺乳动物的植入前胚胎可能容易受到基因组印记机制的破坏，导致印记丢失。这种破坏可能会对胎儿和胎盘的发育产生影响，例如人类的Beckwith-Wiedemann综合征和绵羊的大型后代综合征。我们的目的是调查动态建立单等位基因表达的个人绵羊印迹基因受精后。采用半定量RT-PCR扩增绵羊囊胚、21天胎儿和21天绒毛尿囊的cDNA，比较双亲胚胎和孤雌胚胎之间的表达水平，以指示等位基因表达状态。与其他哺乳动物一样，IGF 2、PEG 1和PEG 3在第21天的孕体中表达为父系，而H19、IGF 2 R、GRB 10和p57 KIP则表达为母系。有趣的是，GNAS在胎儿中表达为母体，但在第21天在绒毛膜尿囊中表达为父体。总体而言，绵羊GRB 10和IGF 2 R的印迹与小鼠相当，但与人不相当。与小鼠和人的滋养层限制性母体表达相反，SASH 2（Mash 2/HASH 2的绵羊同源物）在绵羊胎儿中表达，并在绒毛尿囊中双等位基因表达。绒毛尿囊膜中H19 CTCF III上游区域和IGF 2 R DMR 2的差异甲基化分别主要揭示了父系和母系甲基化，表明这些印记调控区域的保守性。在囊胚中，IGF 2 R、GRB 10和SASH 2呈双等位基因表达，而其他基因未检测到。因此，对于大多数绵羊印记基因的研究，单等位基因的表达不会发生，直到囊胚阶段后。
The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resulting in loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such as Beckwith-Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamics of establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR was used to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels between biparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprinting of ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse and human, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of the H19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation of these imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage.