Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi.
Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi.
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RNAi在小鼠胚胎植入植入术发展中信号通路的功能研究。
DOI：
10.1186/1471-213x-5-28
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发表时间：
2005-12-28
影响因子：
--
通讯作者：
Zernicka-Goetz, M
中科院分区：
文献类型：
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作者：
Soares, ML;Haraguchi, S;Torres-Padilla, ME;Kalmar, T;Carpenter, L;Bell, G;Morrison, A;Ring, CJA;Clarke, NJ;Glover, DM;Zernicka-Goetz, M
小鼠基因功能的研究主要依赖于通过同源重组的基因靶向。然而，这种方法很难应用于特定的时间窗口，并且难以同时敲低多个基因。在这里，我们报告了一种有效的方法，dsRNA介导的基因沉默在晚卵裂期小鼠胚胎，允许检查在植入后阶段的表型。我们表明，通过电穿孔将Bmp4 dsRNA引入完整的胚泡中概括了原肠胚形成时的遗传Bmp4无效表型。这也揭示了Bmp4在调节前内脏内胚层特异性基因表达及其定位中的新作用。我们还表明，RNAi可以用于同时靶向几个基因。当应用于Dishevelled的三种鼠同种型时，其导致比先前在双敲除中观察到的更早的缺陷。这些包括植入后发育严重延迟以及头褶阶段前中线和神经褶的缺陷。我们的研究结果表明，BMP 4信号通路有助于前内脏内胚层的发展，并揭示了小鼠Dishevelled基因产物之间的早期功能冗余。所提出的方法构成了一个强大的工具来筛选基因的功能，管理小鼠胚胎的发育。
Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.