Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepción, Avenida Vicente Méndez 595, Chillán 537, Chile.
Theriogenology. 2010 Jan;73(1):71-85. doi: 10.1016/j.theriogenology.2009.08.003.
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.
本研究旨在鉴定在牛（Bos Taurus）克隆胚胎发育第 17 天（Day 0=核转移或体外受精日）伸长过程中表达的 microRNAs (miRNAs)。通过手工克隆（HMC）或体外受精产生的第 7 天牛扩展囊胚被批量转移到同步受体牛中（48 个 HMC 胚胎到 10 个受体和 28 个体外产生的胚胎到 4 个受体）。在第 17 天回收伸长胚胎；分离 miRNA 并使用定制的复合幻灯片进行微阵列筛选，该幻灯片上点有人类、小鼠和大鼠以及计算机预测的 miRNA。在克隆胚胎和体细胞核供体细胞中确定了表达 miRNA 的初始图谱；在体细胞核转移后，图谱发生了变化，鉴定出克隆和体外生产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）验证了微阵列数据。微阵列和 qPCR 数据之间的相关性为 83%（P=0.01）。基于 qRT-PCR，在克隆牛胚胎中确认了一些供体细胞中的 miRNA 得到了正确的重编程，而其他体细胞 miRNA 则没有得到适当的重编程。一些重编程程度相同的 miRNA 簇位于牛基因组的同一染色体位置上。总之，miRNA 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 miRNA 在哺乳动物植入前发育中的作用具有深远意义。
