Short hairpin RNA and retroviral vector-mediated silencing of p53 in mammalian cells.
RNA interference (RNAi) is the process of gene silencing, in which the recognition of double-stranded RNA ultimately leads to post-transcriptional suppression of gene expression. RNAi induced by small interfering RNA (siRNA) or short hairpin RNA (shRNA) is an important research approach for the analysis of gene function in mammalian cells. Here we established a shRNA expression retroviral vector in which the sense and antisense sequences targeting wild type human p53 were linked together with a 9-nucleotide loop. Further, we found that shRNA expressed from a RNA polymerase III vector based on the human H1 RNA promoter could effectively inhibit gene expression in a sequence-specific manner. H1-driven shRNA dramatically reduced the expression of wild type human p53 in H1299 cells transfected with wtp53. We also found that the retroviral vector could circumvent the difficulty in transfection for the delivery of siRNA or shRNA and the generation of long-term gene silencing. The use of shRNA expression vector for RNAi might provide a rapid and versatile method for assessing gene function in mammalian cells with possible applications in the treatment of diseases.
Liu
Xiao-dong
XD
Radiobiology Research Unit affiliated to Ministry of Health of China, School of Public Health, Norman Bethune Medical Center, Jilin University, Changchun 130021, China. liuxd2002@yahoo.com
Ma
Shum-mei
SM
Liu
Yang
Y
Liu
Shu-zheng
SZ
Sehon
Alec
A
D001483:Base Sequence; D002460:Cell Line; D016158:Genes, p53; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D034622:RNA Interference; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics
D016372:RNA, Antisense; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53