The Construction Of Eukaryotic Expression Vector Of Short Hairpin RNA For CXCR4 And Its Expression In Eukaryotic Cells

Objective: To construct eukaryotic expression vectors of RNA interference specific for human CXCR4 and to detect the effect of recombinant retroviral vectors on CXCR4 mRNA in leukemia cell line (SHI-1) cells, the recombinant retroviral vectors lay the basis for the investigating the function of CXCR4 and searching a new gene therapy method of leukemia.Methods: The mRNA sequence of CXCR4 gene was retrieved from Genbank ,the siRNA (small interfering RNA) was designed according to the Tusch1 principle ,and was converted into complementary DNA coding expression of shRNA (small hairpin RNA). The complementary DNA was synthesized and inserted into retroviral vector(pRNAT-H1.1/Retro), and the recombinant retroviral vectors were identified by enzyme cutting method, Polymerase Chain Reaction(PCR) and the sequence analysis. Then, the vectors were transfected into the packing cell line (PT67) by Lipofectamine 2000; SHI-1 cells were infected by the virus supernatant collected from the PT67 cells, the stable cell lines were selected by G418, the expression of CXCR4 mRNA was assayed by RT-PCR. At last, cell subclones were isolated by limiting dilution.Results: The recombinant plasmids identified by the enzyme cutting method, PCR and the sequence analysis completely coincided with the designs. Compared with SHI-1 cells and SHI-1 cells transfected with negative control plasmid, the SHI-1 cells transfected with interferential expression vector showed a lower CXCR4 mRNA.Conclusion: The shRNA eukaryotic expression vector against CXCR4 mRNA has been successfully constructed, and it effectively inhibits the expression of CXCR4 in SHI-1 cells. It will be a useful tool for investigating the function of CXCR4 gene and for searching a new gene therapy method of leukemia.