Optimization and in vitro validation of EGFP expression controlled by porcine insulin promoter
Received:April 23, 2014  
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DOI:10.3969/j.issn.1005-4847.2014.06.006
KeyWord:Porcine insulin promoter;Specific expression;MIN-6 mouse pancreatic β-cell;EGFP;Wuzhishan miniature pig
              
AuthorInstitution
于淑珍 中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与种质创新重点实验室, 北京 ;四川农业大学动物科技学院, 四川 雅安
冯冲 中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与种质创新重点实验室, 北京
石宁宁 中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与种质创新重点实验室, 北京
宋小凤 中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与种质创新重点实验室, 北京
潘登科 中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与种质创新重点实验室, 北京
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Abstract:
      Objective Getting the robust exogenous gene expression vector under the control of porcine insulin promoter, and to lay the foundation for pancreatic β-cells specific transgene expressing pigs. Method Using porcine insulin promoter (PIP, 1500 bp of the 5'UTR from the porcine INS gene including the first exon and the first intron) to construct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP. Considering that the different location of restriction site may affect the expression efficiency of the transgene, we optimized the expression vector. Firstly the HindIII restriction site was deleted to realize the seamless connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3'intron splicing acceptor site(SA)of the first intron into HindIII restriction site, named as PIP-SA(M)-EGFP. Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells. The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expression efficiency of vectors. Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreatic β-cells. RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with intron splicing. The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing. Mutation of the PIP splice site would cause the first intron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene. Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter. It provides the foundation for preparation of pigs with pancreatic β-cells specifically expressing the transgene.
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