Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing.
RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. One major challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several published bioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical and laborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premise that candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFP signal reduction was closely correlated with siRNA knockdown efficiency of human beta-catenin, as well as with the inhibition of beta-catenin/Tcf4 signaling activity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs via viral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites.
Luo
Qing
Q
The Children's Hospital, and the Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing University of Medical Sciences, Chongqing 400016, China.
Kang
Quan
Q
Song
Wen-Xin
WX
Luu
Hue H
HH
Luo
Xiaoji
X
An
Naili
N
Luo
Jinyong
J
Deng
Zhong-Liang
ZL
Jiang
Wei
W
Yin
Hong
H
Chen
Jin
J
Sharff
Katie A
KA
Tang
Ni
N
Bennett
Erwin
E
Haydon
Rex C
RC
He
Tong-Chuan
TC
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D051176:beta Catenin / Q000235:genetics
C495270:CTNNB1 protein, human; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D051176:beta Catenin; D049452:Green Fluorescent Proteins; D004247:DNA