Construction of Specific Silencing Vector-based siRNA on stathmin Gene Expression in Eca109 Cell Line

Objective 　To construct and identify a recombinant retroviral vector pSUPER-S that target stathmin gene and a stable virus-producing cell line. Methods 　The 64nt encoded targeting stathmin gene shRNA sequence was cloned into a ret roviral vector pSUPER-EGFP with DNA recombinant technique.The recombinant vector was identified by the electrophoresis analysis of rest riction enzyme digestion and DNA sequencing. The packaging cell Eca109 was transfected with this recombinant plasmid using liposome-based transfection and the stable intergrant was selected by using G418 medium. The expression of stathmin mRNA was detected by RT-PCR in t ransfected Eca109 cell lines. Results 　The elect rophoresis of EcoR Ⅰand Hind Ⅲdigested product s showed two DNA fragments, 4692 nt and 285 nt, respectively.The result of sequence demonst rated that 64nt had been inserted into the vector. Green fluorescent protein ( EGFP) was observed after transfection. G418 resistant clones were also selected. Comparing with control groups the expression of stathmin gene was inhibited obviously in t reated group s with pSUPER-S. Conclusion 　A recombinant retroviral vector pSUPER-S that specific silence stathmin gene and a stable virus-producing packaging cell line were successfully constructed and identified.