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| Construction and Identification of Doxycycline-inducible shRNA Expressing Vector Targeting Porcine Nanog |
| ZHANG Li, LUO Yi-bo, MU Yan-shuang, ZHU Jiang, LI Hui, LIU Zhong-hua |
| College of Life Science, Northeast Agricultural University of China, Harbin 150030, China |
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Abstract Since the discovery of RNA interference (RNAi), RNAi has become an important method for studying genes’ function in mammalian cells. RNAi-mediated knockdown or post-transcriptional gene silencing of the gene of interest is achieved either by transfection of synthetic small interfering RNAs (siRNAs) or by expression of short hairpin RNAs (shRNAs). However, if the targeted gene is essential for cell growth and viability, constitutive expression of shRNAs may not be possible or appropriate. Doxycycline-induced gene silencing, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying these genes’ function. Conventional method is to construct binary system to perform inducible gene silencing, including inducible RNA polymeraseⅢ( polⅢ) promoters with embedded tetracycline operators (tetO) and co-expression of the tetracycline regulatory protein, TetR. In the absence of tetracycline or its derivative doxycycline, TetR homodimer binds tightly to the Tet operon(s) inserted within the promoter and acts to suppress transcription. Upon its addition, tetracycline binds with high affinity to the Tet repressor homodimer and causes a conformational change in the repressor that renders it unable to bind to tetO, allowing expression of the shRNA transcript. However, the process for generate stable cell line whose target gene knock-down is time-consuming multi-step process which limited transgenic animals’ expansion. In addition, most shRNA expression units rely on polⅢ promoters such as the H1 or U6 promoter, but it is better to use RNA polymeraseⅡ( polⅡ) promoters for tissue specific gene silencing. To overcome the limitation of shRNA expression systems in mammalian cells, a single vector which only incorporates tetO-hUbc promoter, tTS was used in this study. Firstly, five pGenesil1.0-shRNA plasmids were constructed and selected for the most effective shRNA target on porcine Nanog by Realtime-PCR. In order to generate a single-vector harboring polⅢ promoter allowing for rapid generation of stable cell lines for regulatable shRNA expression targets on porcine Nanog, the selected effective shRNA whose interference efficiency could reach 80% was inserted to a modified doxycycline-induced gene silencing vector TREsilencer. Realtime-PCR analysis demonstrates that efficiency of Nanog silencing could reach about 70%, with increasing concentration of doxycyline. It is a fundamental work for generating Nanog stably silenced porcine cell lines and further studying on Nanog genes’ function.
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Received: 09 May 2011
Published: 25 September 2011
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