Stable Suppression of Gene Expression in Murine Embryonic Stem Cells by RNAi Directed from DNA Vector‐Based Short Hairpin RNA (original) (raw)
Journal Article
College of Life Sciences, Peking University
, Beijing,
P. R. China
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College of Life Sciences, Peking University
, Beijing,
P. R. China
Search for other works by this author on:
College of Life Sciences, Peking University
, Beijing,
P. R. China
Search for other works by this author on:
College of Life Sciences, Peking University
, Beijing,
P. R. China
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Yan Shi ,
College of Life Sciences, Peking University
, Beijing,
P. R. China
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College of Life Sciences, Peking University
, Beijing,
P. R. China
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College of Life Sciences, Peking University
, Beijing,
P. R. China
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Bo Zhang ,
College of Life Sciences, Peking University
, Beijing,
P. R. China
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College of Life Sciences, Peking University
, Beijing,
P. R. China
Correspondence: You-Fang Xue, M.D., Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, 100871, P. R. China. Telephone: 86‐10‐6275‐1858; Fax: 86‐10‐6288‐5710 e-mail: [email protected]
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Published:
01 January 2004
Cite
Fu‐Chou Tang, Guo‐Liang Meng, Hong‐Bo Yang, Cheng‐Jian Li, Yan Shi, Ming‐Xiao Ding, Ke‐Gang Shang, Bo Zhang, You‐Fang Xue, Stable Suppression of Gene Expression in Murine Embryonic Stem Cells by RNAi Directed from DNA Vector‐Based Short Hairpin RNA, Stem Cells, Volume 22, Issue 1, January 2004, Pages 93–99, https://doi.org/10.1634/stemcells.22-1-93
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Abstract
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.
Copyright © 2004 AlphaMed Press
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