miR-122过表达转基因小鼠质粒构建及其功能验证

中国生物工程杂志

2011, Vol. 31

Issue (9): 28-34

研究报告

miR-122过表达转基因小鼠质粒构建及其功能验证

马纪^1,3,4, 孙奋勇², 张越^1,3,4, 洪岸^1,3,4

1. 暨南大学生物工程研究所广州 510632;
2. 上海市第十人民医院上海 200072;
3. 广东省生物工程药物重点实验室广州 510632;
4. 基因工程药物国家工程研究中心广州 510632

The Construction Strategy of miR-122 Overexpression Plasmid in Transgenic Mouse Studies and Its Functional Verification

MA Ji^1,3,4, SUN Fen-yong², ZHANG Yue^1,3,4, HONG An^1,3,4

1. Bioengineering Institute of Jinan University,Guangzhou 510632,China;
2. Shanghai Tenth People’s Hospital,Shanghai 200072,China;
3. Guang Dong Provincial Key Laboratory of Bioengineering Medicine,Guangzhou 510632,China;
4. National Engineering Research Center of Genetic Medicine,Guangzhou 510632,China

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摘要：

目的:比较两种miR-122转基因小鼠过表达载体构建方法,为建立miR-122过表达转基因小鼠奠定基础。方法:PCR扩增长约291bp的pre-miR-122的序列,分别定向克隆到pBROAD3-GFP载体GFP基因上游内含子或下游3'UTR区域,两种质粒分别转染293T细胞,Q-PCR检测miR-122和GFP的表达水平,并观察GFP绿色荧光。miR-122 sensor reporter是将3个miR-122成熟序列的反义序列串联克隆至psiCHECK2载体luciferase 3'UTR中,然后分别与2种miR-122过表达质粒载体共转染293T细胞,最后检测荧光素酶活性来鉴定miR-122调控功能。结果:2种构建方法的miR-122表达水平都明显增高,而只有插入到GFP基因3'UTR的质粒表达GFP功能正常。结论:构建microRNA过表达载体时,microRNA位于报告基因3'UTR区域不会影响microRNA和报告基因的功能;构建的两种miR-122过表达质粒载体都可应用到转基因小鼠研究中,而将miR-122插入到GFP下游的方法则更利于miR-122的表达。

关键词： miR-122; 过表达; Sensor reporter; 转基因小鼠

Abstract:

Objective: In order to establish miR-122 transgenic mouse with GFP, two construction strategies of miR-122 overexpression plasmid were compared. Methods: The pre-miR-122 sequence (291bp) was respectively cloned into the intron upstream or 3'UTR downstream the GFP gene,which was inserted into pBROAD3 vector before. After the two plasmids were transfected into 293T cells, the mRNA expression levels of miR-122 and GFP were detected by Q-PCR, and the fluorescent of GFP in 293T were observed by fluorescence microscope. Another vector, miR-122 sensor reporter, was constructed with three repeated antisense sequences of miR-122 inserted into the 3'UTR of luciferase in psiCHECK2 vector. The regulation function of miR-122 was identified by detecting luciferase activity after the sensor reporter and miR-122 overexpression plasmids were co-transfected into 293T cells. Results: These two construction strategies were proved to increased the expression level of miR-122, but only the plasmid with miR-122 in GFP 3'UTR could express activated GFP. Conclusion: Inserting microRNA into 3'UTR of GFP will not influence both of the expression level and the activity of miR-122 and GFP. The vector pBROAD-GFP-miR-122 is adapted for the study of transgenic mouse.

Key words: miR-122 Overexpression Sensor reporter Tansgenic mouse

收稿日期: 2011-04-25 出版日期: 2011-09-25

ZTFLH:

Q782

基金资助:

国家"973"计划 (2011CBA01006;932004)、黑龙江省教育厅"新世纪"人才基金(1153-NCET-007)资助项目

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引用本文:

马纪, 孙奋勇, 张越, 洪岸. miR-122过表达转基因小鼠质粒构建及其功能验证[J]. 中国生物工程杂志, 2011, 31(9): 28-34.

MA Ji, SUN Fen-yong, ZHANG Yue, HONG An. The Construction Strategy of miR-122 Overexpression Plasmid in Transgenic Mouse Studies and Its Functional Verification. China Biotechnology, 2011, 31(9): 28-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I9/28

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