Stable Suppression of Gene Expression in Murine Embryonic Stem Cells by RNAi Directed from DNA Vector-Based Short Hairpin RNA

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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)93-99
Number of pages7
JournalStem Cells
Volume22
Issue number1
DOIs
StatePublished - 2004

Keywords

  • ES cell
  • Green fluorescent protein
  • RNAi
  • Short hairpin RNA
  • U6 promoter

ASJC Scopus subject areas

  • Molecular Medicine
  • Developmental Biology
  • Cell Biology

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