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Construction and characterization of human Smad3 shRNA expression vector
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 2
- DOI:
- 10.3321/j.issn:1000-2790.2008.02.003
- Key Word:
- Smad3;RNA干扰;遗传载体;肿瘤生长抑制
Abstract： 目的:构建人Smad3的shRNA表达载体,运用RNAi下调Smad3基因在肿瘤细胞系中的表达,观察对肿瘤细胞生长的影响. 方法:化学合成针对人Smad3基因的dsRNA,瞬时转染入Hela细胞,检测RNA干扰效果,筛选有效干扰靶位点;设计并合成针对人Smad3基因的siRNA寡核苷酸链,经退火形成双链后克隆入质粒载体pSilencerTM3.1-H1 hygro,转染入Hela细胞,用hygromycin B筛选稳定单克隆细胞株,对细胞株行RT-PCR和Western Blot检测,观察siRNA对靶基因的抑制效果,进一步研究下调目的分子表达水平以后肿瘤细胞生长的变化. 结果:瞬时转染筛选到了有效的RNA干扰靶位点,构建载体后,建立稳定转染的单克隆细胞株,Smad3 mRNA和蛋白质水平均显著下调,导致肿瘤细胞生长抑制. 结论:成功构建了针对人Smad3高效、特异、作用持久的shRNA表达载体,转染细胞后可下调Smad3的表达,为进一步研究Smad3的功能和肿瘤的基因治疗奠定了基础.
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