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

China Biotechnology

2011, Vol. 31

Issue (9): 28-34 DOI:

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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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

Received: 25 April 2011 Published: 25 September 2011

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Cite this article:

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.

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

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