Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA.
Murine embryonic stem (ES) cells are an ideal system for the research of directed differentiation in vitro. Long double-stranded RNA, which can induce RNA interference (RNAi) effectively in many organisms, has been shown to suppress target gene expression efficiently and specifically in undifferentiated ES cells. However, it cannot be used in differentiated ES cells due to unspecific inhibition of gene expression resulting from the activation of interferon pathway following differentiation. Using green fluorescent protein (GFP) as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into ES cells. Furthermore, when the expression vector is stably integrated into the genome of the cell, it can still show specific RNAi effect, which can be maintained at least for 10 days. These transfected ES cells showed no obvious differences in the morphology or growth rate in culture compared with untransfected cells, suggesting that the activation of shRNA-directed RNAi did not affect the properties of ES cells and that the RNAi effect in ES cells is specific and persistent. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to study the function of genes expressed in ES cells. These ES cells, after integration of the U6-based RNAi vector into their genome, could be used to generate gene knockdown mice.
Tang
Fu-Chou
FC
College of Life Sciences, Peking University, Beijing, P. R. China.
Meng
Guo-Liang
GL
Yang
Hong-Bo
HB
Li
Cheng-Jian
CJ
Shi
Yan
Y
Ding
Ming-Xiao
MX
Shang
Ke-Gang
KG
Zhang
Bo
B
Xue
You-Fang
YF
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007372:Interferons / Q000235:genetics; D008164:Luminescent Proteins; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000235:genetics*; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D014162:Transfection / Q000379:methods
D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; D007372:Interferons