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Stable and Uniform Gene Suppression by Site-Specific Integration of siRNA Expression Cassette in Murine Embryonic Stem Cells

^a Department of Bioscience, National Cardiovascular Center Research Institute;
^b Department of Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, Suita, Osaka, Japan

Key Words. Embryonic stem cells • Green fluorescent protein • Small interfering RNA • Homologous recombination • Hprt locus • Flow cytometry

Correspondence: Takayuki Morisaki, M.D., Ph.D., Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Telephone: 81-6-6833-5012, ext. 2506; Fax: 81-6-6835-5451; e-mail: morisaki{at}ri.ncvc.go.jp

We developed a simple system to introduce small interfering RNA (siRNA) into murine embryonic stem cells (ESCs) and then showed its stable and uniform expression. Using hypoxanthine guanine phosphoribosyl transferase 1 (Hprt)–deficient ESCs as a recipient, we efficiently introduced an siRNA expression cassette into the Hprt locus by homologous recombination, which was easily detected by HAT selection. Nearly all of the HAT-resistant clones exhibited a silenced expression of the exogenous target gene (enhanced green fluorescent protein [EGFP]) or the endogenous target gene (Flk1). Flow cytometry profiles demonstrated that there were no significant differences in level of suppression among individual clones and cells. The suppressing effect by siRNA was maintained for more than 1 month in both undifferentiated and differentiated ESCs, while its persistent expression did not disturb their growth or differentiation potential. The stable and uniform suppression capability of this system will help to screen genes and provide important information regarding cell differentiation in ESCs.