MicroRNA expression profiling of elongated cloned and in vitro-fertilized bovine embryos
MicroRNA expression profiling of elongated cloned and in vitro-fertilized bovine embryos
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DOI：
10.1016/j.theriogenology.2009.08.003
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发表时间：
2010-01-01
期刊：
影响因子：
2.8
通讯作者：
Einspanier, R.
中科院分区：
文献类型：
--
作者：
Castro, F. O.;Sharbati, S.;Einspanier, R.
本研究的目的是鉴定牛（Bos Taurus）克隆胚胎在发育第17天（第0天=细胞核移植或体外受精当天）延长期间表达的microRNA（miRNA）。将通过手工克隆（HMC）或体外受精产生的第7天牛扩增囊胚批量转移至同步受体牛（48个HMC胚胎转移至10个受体，28个体外产生的胚胎转移至4个受体）。在第17天取回伸长的胚胎;分离miRNA，并使用用人、小鼠和大鼠和计算机预测的miRNA点样的定制复合载玻片进行微阵列筛选。在克隆胚胎和体细胞供体细胞中确定表达的miRNA的初始谱;该谱在体细胞核转移后改变，鉴定克隆和体外产生的牛胚胎之间差异表达的miRNA。此外，使用miRNA特异性定量逆转录聚合酶链反应（qRT-PCR）方法（miR-Q）验证微阵列数据。微阵列和qPCR数据之间有83%的相关性（P = 0.01）。基于qRT-PCR，在克隆牛胚胎中证实了来自供体细胞的一些miRNA的正确重编程，而其他体细胞miRNA没有被适当重编程。一些同样重编程的miRNA聚集在牛基因组的相同染色体位置。总之，miRNAs的重编程似乎发生在克隆牛胚胎中。这可能对阐明体细胞克隆中的核重编程以及miRNAs在植入前哺乳动物发育中的作用具有深远的意义。(C)2010年爱思唯尔公司All rights reserved.
The objective of this study was to identify microRNAs (miRNAs) expressed in bovine (Bos Taurus) cloned embryos at Day 17 of development (Day 0 = day of nucleus transfer or in vitro fertilization) during elongation. Day 7 bovine expanded blastocysts produced by hand made cloning (HMC) or in vitro fertilization were bulk-transferred to synchronized recipient cattle (48 HMC embryos to 10 recipients and 28 in vitro produced embryos to four recipients). Elongated embryos were retrieved at Day 17; miRNAs were isolated and subjected to microarray screening using custom composite slides spotted with human, mouse, and rat and in silico predicted miRNAs. An initial profile of expressed miRNAs was determined in cloned embryos and somatic donor cells; this profile changed after somatic cell nucleus transfer, identifying differentially expressed miRNAs between cloned and in vitro produced bovine embryos. Furthermore, microarray data were validated using a miRNA-specific quantitative reverse transcription polymerase chain reaction (qRT-PCR) approach (miR-Q). There was an 83% correlation (P = 0.01) between microarray and qPCR data. Based on qRT-PCR, correct reprogramming of some miRNAs from the donor cells was confirmed in cloned bovine embryos, whereas other somatic miRNAs were not appropriately reprogrammed. Some of the miRNAs that were equally reprogrammed clustered on the same chromosomal location in the bovine genome. In conclusion, reprogramming of miRNAs seemed to occur in cloned bovine embryos. This could have profound implications for elucidating nuclear reprogramming in somatic cloning, as well as for the role of miRNAs in preimplantation mammalian development. (C) 2010 Elsevier Inc. All rights reserved.