猪Nanog基因四环素诱导干扰载体的构建和鉴定

中国生物工程杂志

2011, Vol. 31

Issue (9): 35-42

研究报告

猪Nanog基因四环素诱导干扰载体的构建和鉴定

张力, 罗一博, 牟彦双, 朱江, 李慧, 刘忠华

东北农业大学生命科学院哈尔滨 150030

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

全文: PDF(799 KB) HTML

摘要：

Nanog基因是在早期胚胎和干细胞等多能性细胞中特异表达的重要基因,但有关猪Nanog基因功能的相关研究甚少。四环素诱导干扰载体是一种可通过四环素等药物条件性诱导干扰目的基因的载体,尤其适用于在发育过程中起着关键作用的基因沉默。常规的四环素干扰系统为二元载体,与一元载体相比获得针对特定基因干扰的稳定细胞系所需周期更长。首先通过构建pGenesil 1.0-shRNA重组干扰载体,瞬时转染稳定过表达猪Nanog基因的猪胎儿成纤维细胞后通过Realtime-PCR筛选出干扰效率可达80%以上的干扰片段。之后将筛选得到的干扰片段插入到改造的一元四环素诱导干扰载体TREsilencer,对稳定表达猪Nanog基因的猪胎儿成纤维细胞进行了瞬时转染。实验分别通过光密度检测以及Realtime-PCR检测了不同浓度doxycycline的诱导效率和干扰效率。结果表明,所构建的四环素诱导干扰载体TREsilencer-shRNA5随着四环素浓度的增加,诱导Nanog基因的干扰效率增加,在处理浓度为1μg/ml时干扰效率可达70%以上,为后续得到可诱导的稳定干扰猪Nanog基因的细胞系和进一步研究猪Nanog基因功能奠定了基础。

关键词： 四环素; 诱导; 干扰; Nanog; 猪

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.

Key words: Doxycyline Inducible Gene silencing Nanog Pig

收稿日期: 2011-05-09 出版日期: 2011-09-25

ZTFLH:

Q786

基金资助:

北京市科委资助项目(Z0005187040621)

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引用本文:

张力, 罗一博, 牟彦双, 朱江, 李慧, 刘忠华. 猪Nanog基因四环素诱导干扰载体的构建和鉴定[J]. 中国生物工程杂志, 2011, 31(9): 35-42.

ZHANG Li, LUO Yi-bo, MU Yan-shuang, ZHU Jiang, LI Hui, LIU Zhong-hua. Construction and Identification of Doxycycline-inducible shRNA Expressing Vector Targeting Porcine Nanog. China Biotechnology, 2011, 31(9): 35-42.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I9/35

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