Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.
Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.
复制标题
在体外培养的植入前小鼠胚胎中通过反义吗啉寡核苷酸敲低基因表达。
DOI：
10.1016/j.ab.2016.06.032
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发表时间：
2016
影响因子：
2.9
通讯作者：
Sasaki A. and Kobayashi M.
中科院分区：
文献类型：
--
作者：
Sato Y.;Sato S.;Kikuchi T.;Nonaka A.;Kumagai Y.;Sasaki A. and Kobayashi M.
反义吗啉寡核苷酸（MOs）敲低基因表达是分析哺乳动物细胞基因功能的一种简单有效的方法。在此，我们证明了一种用于MOs的特殊转染试剂Endo-Porter（EP）将MOs有效地递送到体外培养的植入前小鼠胚胎中。应用荧光素标记的对照MO来监测在不同量的EP和牛血清白蛋白存在下MO掺入发育中的2-细胞胚胎中。在优化的条件下，荧光检测在2-细胞胚胎在3小时的孵育期内。为了分析优化条件的有效性，应用反义Oct 4 MO敲低2-细胞期发育胚胎中OCT 4蛋白的合成。在胚泡中，反义Oct 4 MO诱导OCT 4蛋白的量减少到不到一半。还观察到胚泡生长物中几乎完全不存在OCT 4阳性细胞，并且内细胞团几乎完全消失。这些表型与Oct 4缺陷小鼠胚胎的表型一致。总之，我们认为使用EP递送MO对于体外培养的植入前小鼠胚胎中基因表达的敲低是有用的。
Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos culturedin vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisenseOct4MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisenseOct4MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those ofOct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos culturedin vitro.