Evaluation of RNA interference in developing porcine granulosa cells using fluorescence reporter genes
Evaluation of RNA interference in developing porcine granulosa cells using fluorescence reporter genes
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DOI：
10.1262/jrd.50.599
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发表时间：
2004-10-01
影响因子：
1.8
通讯作者：
Hattori, M
中科院分区：
文献类型：
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作者：
Hirano, T;Yamauchi, N;Hattori, M
使用小干扰RNA（siRNA）的基因沉默可能是有用的，在细胞分化过程中表达的未知基因的功能分析。本研究采用两种荧光报告基因：一种编码绿色荧光蛋白（GFP）的质粒和一种编码红色荧光蛋白（RFP）的质粒，对牛卵泡刺激素（FSH）刺激的猪颗粒细胞进行RNA干扰（RNAi）研究。通过脂质体转染将具有两种质粒的靶向GFP mRNA序列的siRNA（GFP-siRNA）引入培养的细胞中。在荧光显微镜下观察GFP和RFP表达细胞，并通过流式细胞术进行分析。在将两种质粒引入细胞后观察到强荧光。GFP产生的绿色荧光的强度被GFP-siRNA的引入大大抑制，显示绿色与红色荧光的比率降低约70%。因此，我们的结论是，基因沉默的siRNA可以用来分析在猪颗粒细胞分化过程中的目的基因的功能。
Gene silencing using small interfering RNA (siRNA) may be useful for functional analyses of unidentified genes expressed during cell differentiation. The present study was performed to evaluate RNA interference (RNAi) in porcine granulosa cells stimulated with bovine FSH, by using two fluorescence reporter genes: a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescence microscopy and analyzed by flow cytometry. Strong fluorescence was observed after introduction of both plasmids into cells. The intensity of green fluorescence generated by GFP was greatly suppressed by introduction of GFP-siRNA, showing an approximate 70% decrease in the ratio of green to red fluorescence. Consequently, we concluded that gene silencing by siRNA can be used to analyze the functions of genes of interest during differentiation of porcine granulosa cells.