A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens
A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens
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DOI：
10.1093/nar/gkh238
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发表时间：
2004-02-01
影响因子：
14.9
通讯作者：
Sellers, WR
中科院分区：
文献类型：
--
作者：
Hsieh, AC;Bo, RH;Sellers, WR
通过RNA干扰（RNAi）进行基因沉默已被确立为进行反向遗传研究的一种手段。为了更好地了解用于高通量细胞筛选的短干扰RNA（SiRNA）敲除的决定因素，选择并合成了148个靶向PI 3 K途径中30个基因的SiRNA双链体。通过定量实时PCR测量22个基因的RNA敲低程度。与有效敲低相关的参数的分析显示：（1）靶向编码序列中间的双链体的沉默显著较差，（ii）靶向3'UTR的双链体的沉默与靶向编码序列的双链体相当，（iii）每个基因汇集四个或五个双链体在敲低基因表达方面是非常有效的，并且（iv）在达到>70%在mRNA的敲低中，在特定位置，最显著的是siRNA双链体的位置11（G或C）和19（T）处存在强烈的核苷酸偏好。最后，在一个原理验证途径的细胞遗传筛选中，进行了检测Akt S473磷酸化的负性遗传调节剂，发现了已知的这种磷酸化的负性调节剂，PTEN和PDKA，这些数据有助于为哺乳动物细胞中的全基因组siRNA筛选奠定基础。
Gene silencing through RNA interference (RNAi) has been established as a means of conducting reverse genetic studies. In order to better understand the determinants of short interfering RNA (siRNA) knockdown for use in high-throughput cell-based screens, 148 siRNA duplexes targeting 30 genes within the PI3K pathway were selected and synthesized. The extent of RNA knockdown was measured for 22 genes by quantitative real-time PCR. Analysis of the parameters correlating with effective knockdown showed that (1) duplexes targeting the middle of the coding sequence silenced significantly poorer, (ii) silencing by duplexes targeting the 3' UTR was comparable with duplexes targeting the coding sequence, (iii) pooling of four or five duplexes per gene was remarkably efficient in knocking down gene expression and (iv) among duplexes that achieved a >70% knockdown of the mRNA there were strong nucleotide preferences at specific positions, most notably positions 11 (G or C) and 19 (T) of the siRNA duplex. Finally, in a proof-of-principle pathway-wide cell-based genetic screen, conducted to detect negative genetic regulators of Akt S473 phosphorylation, both known negative, regulators of this phosphorylation, PTEN and PDKA, were found. These data help to lay the foundation for genome-wide siRNA screens in mammalian cells.