Molecular cloning and functional identification of ST8SiaV promoter

Objective: To clone ST8SiaV promoter,construct ST8SiaV gene promoter-pGL3-Basic-GFP expressing vector and identify the constructed vector. Methods:① Based on the ST8SiaV rat genomic DNA sequence obtained from NCBI by BLAST software, a 1.7 kb targeted sequence from ATG original code was amplified by PCR method.② The product of PCR was inserted into pMD 18-T vector then transferred into E.coli DH5α. The positive clone was picked out and identified by DNA sequencing.③ The identified target promoter was digested with SacⅠ and Bgl Ⅱ, and then inserted into pGL3-Basic plasmid vector. In order to study this gene expressed in vivo during the differentiation of neural stem cells, the luciferase report gene in pGL3-Basic was replaced by GFP.④The recombinant plasmid was transfected into SD neural stem cell to identify the biologic activity.Results and Conclusion: The 1.7 kb 5′flanking region of ST8SiaV gene with activity was successfully cloned and the promoter-pGL3-Basic-GFP vector for studying in vivo was well constructed, laying a foundation for further research into the function of the ST8SiaV gene promoter.