Identification Of Negative Regulatory Element In SLC22A3 Gene

Objective:To localize the key negative regulatory element which is in the intron7 of SLC22A3 gene on chromosome 6q26–q27.Methods:The wild type truncated plasmids of intron7 were constructed by gene recombination to stepwise locate the specific sequence position of this negative regulatory element. Specific truncated gene fragments were amplified the by four stages using bacterial artificial chromosome(BAC),including human SLC22A3 gene as templates, and were inserted into luciferase report vector to construct recombinant plasmids respectively.Recombinant plasmids and internal reference plasmids were cotransfected into HEK293 T cells with ratio of 50:1. The luciferase activity was detected after 24 hours to determine which inserted fragment showed regulatory effects on gene expression, the m RNA level of Luciferase gene was detected by Real time quantitative PCR, and the correlation between the regulatory element and its position was identified by us. At the same time,we predicted the transcription factor binding sites in the element.Results:The luciferase activities of recombinant plasmids of p GL3-proSLCi7-NO6, p GL3-pro-SLCi7-NO10, p GL3-pro-SLCi7-NO6.2, p GL3-pr o-SLCi7-NO6.3, p GL3-pro-SLCi7-NO10.2, p GL3-pro-SLCi7-NO6.21, p GL3-pro-SLCi7-NO10.22 were decreased compared with those of p GL3-Promoter(P<0.05);but there was no significant difference between p GL3-pro-SLCi7-NO6.21-300bp(P>0.05),p GL3-pro-SLCi7-NO10.22-300 b p(P>0.05)and p GL3-Promoter.The level of m RNA was accordant with the protein through Real time quantitative PCR,and the corelation be tween the regulatory elements and its position was independent. At la st, we found there were four transcription factor binding sites in the element.Conclusion:The results demonstrated that the intron7 of SLC22A3 gene on chromosome6q26–q27 contains two negative regulatory elements, which might provide theoretical basis for the further studies at its protein level.