Wang Zhen,Wang Lin,Wan Qianxue,Jin Xing,Liang Guangping,Peng Xi.Construction of a luciferase reporter vector containing hITF promoter[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(10):1024-1027.
Wang Zhen,Wang Lin,Wan Qianxue,Jin Xing,Liang Guangping,Peng Xi.Construction of a luciferase reporter vector containing hITF promoter[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(10):1024-1027.DOI:
Construction of a luciferase reporter vector containing hITF promoter
Objective To construct a luciferase reporter vector containing human intestinal trefoil factor (hITF) promoter and to evaluate its activity in LS-174T and HEK293 cells. Methods A 2.5 kb fragment spanning the region -2 331 to +53 of hITF gene was amplified from genomic DNA extracted from LS-174T by PCR. Then 10 fragments containing different region of the promoter were substantially inserted into promoter-less pGL-3 basic luciferase reporter vector
and the obtained vectors were transfected into HEK-293 cells. In 48 h after the transfection
the cells were lysed and the luciferase activity was measured. Results The inserted hITF promoter was verified by DNA sequencing. The promoter activity of -500/+53
-400/+53 and -300/+53 ranged from 0.023 to 0.026
while that of -200/+53
-100/+53 and -50/+53 from 0.003 2 to 0.004 2. The minimal functional promoter was located in the -300/+53
and there were some potential active elements. The promoter region of -300/-200 played a vital role in transcription initiation of hITF. Conclusion The recombinant plasmid containing luciferase reporter gene driven by hITF promoter is constructed successfully. The region spanning -300/-200 plays an important role in facilitating the initiation of transcription.