Objective: To clone 5' untranslated region of human IPO8 gene and determine its transcription activity.
Methods: We used 5' rapid amplification of cDNA ends (RACE) analysis to identify the IPO8 transcription start site (TSS), and amplified series truncated 5' UTR fragment containing transcription start site. The PCR productions were inserted into luciferase report vector pGL3- Basic. After confirmation by restriction enzyme digestion, the recombinant plasmids were cotransfected into Saos-2 cells with plasmid pRL-TK. The luciferase activities were measured by dual luciferase reporter system.
Results: The IPO8 gene transcription start site was established. The electrophoresis analysis of restriction enzyme digestion and DNA sequencing verified the fragments were successfully amplified and inserted into pGL3-Basic. After the recombinant plasmids transfected, the highexpressions of luciferase were detected in Saos-2 cells.
Conclusion: The recombinant vector containing IPO8 promoter is constructed successfully, which provides a foundation for determining expressional regulation of IPO8 in the further study.