Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairpin RNA.
RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA (shRNA) transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vector-mediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, H1-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.
Hao
De-Long
DL
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
Liu
Chang-Mei
CM
Dong
Wen-Ji
WJ
Gong
Huan
H
Wu
Xue-Song
XS
Liu
De-Pei
DP
Liang
Chih-Chuan
CC
D002460:Cell Line; D005786:Gene Expression Regulation / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D034622:RNA Interference; D016337:RNA, Catalytic / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency* / Q000235:genetics*
D016337:RNA, Catalytic; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; C418481:hairpin ribozyme