基础型无启动子基因打靶载体pMCS-IEN的构建及初步检测

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Abstract Gene targeting is a new molecular biology technique, which introduces heritable genetic modification on the basis of DNA homologous recombination. Compared with classic approaches, homologous recombination using a promoter trap vector could yield higher targeting frequencies. It is very important for the correct expression of exogenous gene. In this study, neor gene was amplified by PCR technology from plasmid pIRES2-EGFP and BamHⅠ and BglⅡ restriction enzyme sites were added to the upstream and downstream, respectively; then the PCR fragment was cloned into pMD19-T simple Vector, named pMD-neo. Plasmids pMD-neo and pEGFP-C1 were digested by BamHⅠ and BglⅡ, and ligated together to obtain intermediate plasmid pEGFP-neo. Then pEGFP-neo and pIRES2-EGFP were digested by Bsp1407Ⅰ and SfiⅠto constitute another intermediate plasmid pIRES-EGFPneo. At last, multiple cloning sites (MCS) were added to pIRES-EGFPneo after digestion of pIRES-EGFPneo and pMD-MCS with BamHⅠand MluⅠ, and the final gene trap vector pMCS-IEN was constituted. To test the expression of green fluorescence protein (GFP) and G418 resistance gene, the PGK promoter was added to the upstream of MCS of the pMCS-IEN and the reconstitute plasmid was transfected to Hela cells. GFP and G418 resistance colonies were obtained after screening by microscopy and G418 selection. When the homologous arms were inserted into the basic vector pMCS-IEN and formed the promoter trap vector，it could be used to target the objective gene. Therefore, this vector will become a useful tool to generate gene modification animals.

Key words： Gene targeting Promoter trap vector Fusion protein Transfection Green fluorescence protein (GFP)

Received: 04 December 2014 Published: 13 May 2015

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	LIANG Gao
	LIU Hui
	HAN Xue-Ji
	DIAO Yu-Hang
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	YUN Ting
	LIU Dong-Jun
	XU Ri-An
	LI Rong-Feng
	LI Xue-Ling

Cite this article:

LIANG Gao,LIU Hui,HAN Xue-Ji, et al. Construction and Preliminary Test of the Basic Promoter Trap Vector pMCS-IEN for Gene Targeting[J]. , 2015, 23(7): 973-980.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2015/V23/I7/973

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