Construction and identification of eukaryotic expression vector expressing double shRNA sections targeting heparanase gene

    LIU Yu, XIN Xiaoª²Yan, MAO Jing, ZHANG Wei

    Department of Obstetrics and Gynecology, Xijing Hospital£¬ Fourth Military Medical University, Xi¬ðan 710033£¬ China

    ¡¾Abstract¡¿ AIM: To construct and identify eukaryotic expression vector expressing double shRNA sections targeting heparanase gene (Hpaª²shRNA). METHODS: Six pairs of oligonucleotides were designed and chemically synthesized. After randomly connecting an oligonucleotide and another one with 4-8 bases pairs interval, they were directionally inserted into plasmid pGenesilª²1 with respectively U6 promoter and termination code, the common green fluorescence protein (EGFP) gene and Neo gene. In this way, 3 vectors of pGenesilª²1ª²Hpaª²shRNA containing 2 sections of Hpaª²shRNA were constructed and they were transfected into the ovarian cancer cell SKOV3. The rate of transfection was detected by flow cytometry and fluorescence microscope. The inhibition effectiveness of Hpa protein was analyzed by immunohistochemical staining. RESULTS: The constructed eukaryotic expression vectors effectively suppressed the Hpa expression in transfected cells. The 3 vectors of different short hairpin RNA of Hpa effectively suppressed the expression in SKOV3(P<0.01). CONCLUSION: We have constructed a eukaryotic expression vector of shRNA, specific for Hpa and have constructed and identified a eukaryotic expression vector of doubleª²gene short hairpin RNA for Hpa. ......