Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system
Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system
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DOI：
10.1002/mrd.20047
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发表时间：
2004-05-01
影响因子：
2.5
通讯作者：
Seto, A
中科院分区：
文献类型：
--
作者：
Haraguchi, S;Saga, Y;Seto, A
近年来，小干扰RNA（siRNA）已成为分析哺乳动物细胞基因功能的有力工具。在这里，我们报告，显微注射siRNA表达载体到细胞核中是一种有效和强大的方法，在植入前小鼠胚胎的特定基因沉默。我们用这种方法检测了两个基因EGFP和Oct 4的表达。将编码靶向EGFP或Oct 4的siRNA的载体注射到受精卵的原核或核中，然后将其培养至胚泡阶段。当在胚泡期卵中检查RNAi的作用时，在mRNA和蛋白质水平上以浓度依赖性方式对基因产物产生强烈抑制。其他内源基因的表达不受影响，显示了载体介导的RNAi的特异性。此外，该方法对抑制母体表达的mRNA是有效的。为了证明Oct 4的RNAi诱导了与Oct 4缺失胚胎相似的表型，在ES培养基中进一步培养囊胚。在培养的第四天之后，胚胎或者仅生长出一层滋养层细胞，或者在胚泡阶段显示出发育停滞（>90%）。此外，伴随着在胚泡阶段的Oct 4抑制，我们观察到Fgf 4的抑制，Fgf 4是已知在Oct 4表达下游诱导的基因。综上所述，这些结果表明，使用siRNA表达载体是实现植入前胚胎中基因沉默的有效方法。(C)2004 Wiley-Liss，Inc.
Recently, small interfering RNAs (siRNAs) have become a powerful and widely used tool for the analysis of gene function in mammalian cells. Here we report that the microinjection of an siRNA expression vector into the nucleus is an efficient and powerful method of specific gene silencing in preimplantation mouse embryos. We used this method to examine the expression of two genes EGFP and Oct4. Vectors encoding siRNAs targeted against EGFP or Oct4 were injected into the pronucleus or nucleus of zygotes, which were then cultured until the blastocyst stage. When the effects of RNAi were examined in blastocyst stage eggs, there was robust inhibition of the gene product in a concentration-dependent manner at both the mRNA and the protein level. The expression of other endogenous genes was not affected, showing the specificity of the vector-mediated RNAi. In addition, this method was effective for inhibiting maternally expressed mRNA. To demonstrate that RNAi of Oct4 induced a similar phenotype to that of Oct4-null embryos, the blastocysts were further cultured in ES medium. After the fourth day of culture, the embryos either had outgrown only a layer of trophoblast cells or showed developmental arrest at the blastocyst stage (>90%). Moreover, concomitant with Oct4 suppression at the blastocyst stage, we observed inhibition of Fgf4, a gene that is known to be induced downstream of Oct4 expression. Taken together, these results demonstrate that the use of siRNA expression vector is a powerful way to achieve gene silencing in the preimplantation stage embryo. (C) 2004 Wiley-Liss, Inc.