Cell-Free Extracts from Mammalian Oocytes Partially Induce Nuclear Reprogramming in Somatic Cells
Cell-Free Extracts from Mammalian Oocytes Partially Induce Nuclear Reprogramming in Somatic Cells
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DOI：
10.1095/biolreprod.108.073676
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发表时间：
2009-05-01
影响因子：
3.6
通讯作者：
Imai, Hiroshi
中科院分区：
文献类型：
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作者：
Miyamoto, Kei;Tsukiyama, Tomoyuki;Imai, Hiroshi
核移植被认为是研究卵母细胞对哺乳动物体细胞核重编程的唯一可靠工具。然而，核移植不太适合重编程分子机制的生化分析。来自卵母细胞的无细胞系统是模拟体外重编程的一种有吸引力的替代方法，因为可以处理和分析大量细胞。然而，尚未在哺乳动物中开发出使用卵母细胞的无细胞系统。在这里，制备了猪卵母细胞的细胞提取物，并评估了它们诱导核重编程的能力。中期 II (MII) 卵母细胞的提取物消除了可逆透化体细胞中调节基因表达的机制。例如，提取物导致组蛋白脱乙酰化和 TATA 盒结合蛋白从细胞核中消失。然而，MII 提取物处理的细胞在细胞培养后没有表现出任何明显的变化。相比之下，生囊 (GV) 卵母细胞提取物激活多能标记基因，尤其是 NANOG，并在细胞培养后诱导部分去分化。 GV 提取物对多能标记基因的激活与提取物处理过程中诱导的组蛋白乙酰化有关。这些结果表明 GV 和 MII 卵母细胞提取物对核重编程具有不同的作用。此外，两种卵母细胞提取物均诱导 NANOG 上游区域的位点特异性去甲基化。这些结果表明，GV 和 MII 卵母细胞的无细胞提取物可用于研究核重编程机制。
Nuclear transfer has been regarded as the only reliable tool for studying nuclear reprogramming of mammalian somatic cells by oocytes. However, nuclear transfer is not well suited for biochemical analyses of the molecular mechanisms of reprogramming. A cell-free system from oocytes is an attractive alternative way to mimic reprogramming in vitro, since a large number of cells can be treated and analyzed. Nevertheless, a cell-free system using oocytes has not been developed in mammals. Here, cell extracts from porcine oocytes were prepared and their ability to induce nuclear reprogramming was evaluated. Extracts from metaphase II (MII) oocytes erased the machinery for regulating gene expression in reversibly permeabilized somatic cells. For example, the extracts caused histone deacetylation and the disappearance of TATA box-binding protein from the nuclei. However, MII-extract-treated cells did not show any obvious changes after cell culture. In contrast, extracts from germinal vesicle (GV) oocytes activated pluripotent marker genes, especially NANOG, and induced partial dedifferentiation after cell culture. The activation of pluripotent marker genes by GV extracts was associated with histone acetylation that was induced during extract treatment. These results indicate that GV- and MII-oocyte extracts have different roles on nuclear reprogramming. Furthermore, both oocyte extracts induced site-specific demethylation in the upstream region of NANOG. These results indicate that cell-free extracts derived from GV- and MII-oocytes could be useful for studying the mechanisms involved in nuclear reprogramming.