Identification of negative regulatory element in SLC22A3 gene

Abstract:

Objective：To research and identify the specific sequence position of the key negative regulatory element in intron7 of SLC22A3 gene on chromosome 6q26-q27. Methods：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（including human SLC22A3 gene） as templates，and were inserted into luci-ferase report vector to construct recombinant plasmids respectively. Recombinant plasmids and internal reference plasmids were co-transfected into HEK293T cells with ratio of 50∶1，and the luciferase activity was detected after 24 h to determine which inserted frag－ment showed regulatory effect on gene expression；transcription factor binding sites were predicted in element. Results：The luciferase activities of recombinant plasmids of pGL3-pro-SLCi7-NO6，pGL3-pro-SLCi7-NO10，pGL3-pro-SLCi7-NO6.2，pGL3-pro-SLCi7-NO6.3，pGL3-pro-SLCi7-NO10.2，pGL3-pro-SLCi7-NO6.21，pGL3-pro-SLCi7-NO10.22 were decreased compared with those of pGL3-Promoter（P<0.05）；but there was no significant difference in luciferase activity between pGL3-pro-SLCi7-NO6.21-300 bp（P >0.05），pGL3-pro-SLCi7-NO10.22-300 bp（P>0.05）and pGL3-Promoter. Conclusion：The results demonstrated that intron7 of SLC22A3 gene on chromosome 6q26-q27 contains two negative regulatory elements，which might provide theoretical basis for further studies at protein level.