Effects of Donor Fibroblasts Expressing OCT4 on Bovine Embryos Generated by Somatic Cell Nuclear Transfer
Effects of Donor Fibroblasts Expressing OCT4 on Bovine Embryos Generated by Somatic Cell Nuclear Transfer
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DOI：
10.1089/cell.2012.0055
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发表时间：
2013-02-01
影响因子：
1.6
通讯作者：
Cibelli, Jose B.
中科院分区：
文献类型：
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作者：
Goissis, Marcelo D.;Suhr, Steven T.;Cibelli, Jose B.
通过体细胞核移植（SCNT）生产健康、活的克隆动物一直受到低效率的阻碍。显着的表观遗传变化必须发生，以确保适当的染色质重塑SCNT。我们假设OCT 4在与去核卵母细胞融合之前在供体成纤维细胞中的外源性表达将促进SCNT重编程。我们用仅含有黄色荧光蛋白（YFP）或与YFP融合的OCT 4基因（YO）的逆转录病毒载体感染牛成年成纤维细胞。我们发现NT-YFP和NT-YO组之间发育到囊胚阶段没有差异。NT-YFP胚胎具有最少的滋养层细胞，通过CDX 2阳性细胞的数量来测量。表达OCT 4的成纤维细胞具有降低水平的组蛋白3赖氨酸9或27三甲基化（分别为H3 K9 me 3和H3 K27 me 3）。NT-YO囊胚显示出比IVF和NT-YFP胚胎更高的H3 K9 me 3水平;然而，它们没有不同的H3 K27 me 3水平。与体外受精囊胚相比，NT-YO和NT-YF中XIST mRNA的表达水平更高。我们观察到SOX 2、NANOG和CDX 2的表达没有差异。虽然OCT 4在供体细胞中的过表达增加了H3 K9 me 3，并没有减少XIST基因的表达，我们表明，一个单一的转录因子可以影响牛SCNT胚胎滋养外胚层细胞的数量。
The production of healthy, live, cloned animals by somatic cell nuclear transfer (SCNT) has been hampered by low efficiencies. Significant epigenetic changes must take place to ensure proper chromatin remodeling in SCNT. We hypothesized that exogenous expression of OCT4 in donor fibroblasts prior to its fusion with enucleated oocytes would facilitate SCNT reprogramming. We infected bovine adult fibroblasts with retroviral vectors containing yellow fluorescent protein (YFP) only, or the OCT4 gene fused to YFP (YO). We found that development to the blastocyst stage was not different between NT-YFP and NT-YO groups. NT-YFP embryos had the fewest trophoblast cells, measured by numbers of CDX2-positive cells. Fibroblasts expressing OCT4 had reduced levels of histone 3 lysine 9 or 27 trimethylation (H3K9me3 and H3K27me3, respectively). NT-YO blastocysts displayed higher H3K9me3 levels than IVF and NT-YFP embryos; however, they did not have different H3K27me3 levels. Levels of XIST mRNA expression in NT-YO and NT-YF were higher when compared to in vitro-fertilized blastocysts. We observed no differences in the expression of SOX2, NANOG, and CDX2. Although overexpression of OCT4 in donor cells increased H3K9me3 and did not reduce XIST gene expression, we show that a single transcription factor can affect the number of trophectoderm cells in bovine SCNT embryos.