Application of RNA interference to chicken embryos using small interfering RNA.
Application of RNA interference to chicken embryos using small interfering RNA.
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DOI：
10.1002/jez.a.99
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发表时间：
2004-10
期刊：
影响因子：
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通讯作者：
Fuminori Sato;Tetsuto Nakagawa;Makoto Ito;Y. Kitagawa;M. Hattori
中科院分区：
文献类型：
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作者：
Fuminori Sato;Tetsuto Nakagawa;Makoto Ito;Y. Kitagawa;M. Hattori
利用小干扰RNA （siRNA）进行基因沉默在鸟类中尚未建立。采用编码绿色荧光蛋白（GFP）的质粒和编码红色荧光蛋白（RFP）的质粒双荧光报告试验，对鸡胚中的RNA干扰（RNAi）进行了评价。将两种质粒靶向GFP mRNA序列的siRNA （GFP-siRNA）分别通过脂质体转染和微电穿孔转染到培养细胞和全胚中。荧光显微镜下观察GFP和rfp表达的细胞和胚胎，流式细胞仪分析其mrna，逆转录- pcr （RT-PCR）分析其mrna表达。将这两种质粒导入细胞，观察到强烈的荧光。通过引入GFP- sirna， GFP产生的绿色荧光强度被大大抑制。RT-PCR分析显示，引入GFP- sirna也降低了GFP mRNA水平。与GFP相比，RFP产生的红色荧光强度和RFP mRNA水平保持不变。在全胚中，引入GFP-siRNA也能特异性抑制GFP的表达，且抑制持续时间至少为72小时。由此可见，利用siRNA进行基因沉默可用于分析禽类胚胎发生过程中相关基因的功能。
Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.