Cloning of rat SLC7a8 gene and construction of eukaryotic expression vector

Objective: To clone rat SLC7a8 gene cDNA and construct its eukaryotic expression vector for its function study in SHR.Methods: Total RNA was extracted from rat kidney tissue.SLC7a8 cDNA was prepared by RT-PCR,and inserted into pGEM-T Easy vector and confirmed by sequence analysis.Then the pGEM-T Easy vector which contained SLC7a8 cDNA was also amplified by PCR,and cloned into pcDNA3.1(+) plasmid.The recombinant plasmid was confirmed by PCR,double enzyme digestion analysis and DNA sequencing.Then the recombinant plasmid pcDNA3.1(+)-SLC7a8 was transiently transfected into NRK-52E cells using Lipofectamin tm 2 000.The level of expression of SLC7a8 mRNA and protein was identified by RT-PCR and Western blotting after transfection.Results: The cDNA of SLC7a8 was cloned,and its recombinant eukaryotic expression vector was constructed successfully.Over-expression of SLC7a8 mRNA and protein was observed in NRK-52E cells transfected with SLC7a8 gene as compared to the blank control cells and the cells transfected with an empty vector(both P 0.05).Conclusion: The pcDNA3.1(+)-SLC7a8 expression vector was successfully constructed and over-expression of SLC7a8 gene in NRK-52E cells was obtained.The recombinant eukaryotic expres-sion vector will be used to investigate the biologic role of SLC7a8 in hypertension.