Construction of Red Fluorescent Protein Driven by Mouse FABP4 Promoter and Expression in Bovine Cells

doi:10.7668/hbnxb.2016.06.005

Abstract

Abstract: The promoter/enhancer cassette of mouse adipocyte-type fatty acid binding protein(FABP4) was widely used as the adipocyte specific element.To detect the effect of mFABP4 promoter on gene expression in bovine cells,5.9 kb mFABP4 promoter fragment was cloned from mouse liver genome and identified by digestion and sequencing after ligated to pMD19-T vector.By digestion of enzyme EcoT 22I the non-core promoter fragment was deleted from the 5.9 kb fragment and another 2.3 kb core promoter fragment was obtained.The 5.9 kb and 2.3 kb fragment were inserted into the multiple cloning site of plasmid pDs-Red 2-1 by enzyme Sac Ⅱ,respectively.As a result the vectors,pMF5.9-Red and pMF2.3-Red,were constructed successfully.By lipofection,the constructed pMF5.9-Red and pMF2.3-Red vectors were transfected into bovine adipose-derived stem cells and bovine embryo fibroblast cells,respectively.24 h after transfection,Real-time PCR was applied to detect the transcription of red fluorescent protein in transfected cells.Results showed that the sequence of cloned 5.9 kb fragment was correct,and the 5.9 kb and the 2.3 kb fragments were correctly ligated into vectors.The mRNA of red fluorescent protein was both detected in transfected bovine adipose-derived stem cells and bovine embryo fibroblast cells,and the expression level displayed 2 fold higher in pMF2.3-Red than that of pMF5.9-Red.In conclusion,this study constructed the expression vector pMF5.9-Red and pMF2.3-Red with 5.9 kb or 2.3 kb fragment of mouse FABP 4 gene as promoter region could initiate the transcription of foreign genes in bovine cells and the transcription efficiency by 2.3 kb promoter fragment was higher than that of 5.9 kb promoter fragment.

Key words: Mouse FABP4 promoter, Vector construction, Cattle cells

CLC Number:

Q782

YUE Yongli, YU Haiquan. Construction of Red Fluorescent Protein Driven by Mouse FABP4 Promoter and Expression in Bovine Cells[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(6): 26-30. doi: 10.7668/hbnxb.2016.06.005.

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