Construction of Recombinant Retroviral Vector of siRNA Specific for Her-2 and the Expression of Recombinant Vector in SKOV3

Objective 　To establish the stable SKOV3 cell lines with a persistent knockdown of Her-2 by transfecting the recombinant ret roviral vectors of siRNA specific for Her-2 into SKOV3 cell line. Methods 　We synthesized oligonucleotides for Her-2 in vitro, and cloned them into ret roviral vector RNAiReady pSIREN-Retro-Q. Subsequently the plasmids were sequenced and digested to identify the successful construction of the recombinant retroviral vectors. The vectors-based RNAi were transfected into packing cell line PT67, which was selected by puromycin later. SKOV3 was infected by the virus supernatant of stable PT67 cell lines, and the stable SKOV3 cell lines with a persistent knockdown of Her-2 were established by selecting with puromycin, many of which showed significantly reduced levels of Her-2 mRNA and p185 by RT-PCR and immumohistochemistry methods, compared to wild type SKOV3 cells. Results 　The recombinant retroviral vectors were const ructed successfully, and the stable SKOV3 cell lines with a persistent knockdown of Her-2 were established by the infection of virus supernatant secreted by the new stable PT67 cell lines. Conclusion 　The establishment of stable SKOV3 cell lines with a persistent knockdown of Her-2 contributes to the study of the changes of malignant biological activity of tumor cell lines by t ransfection of siRNA ret roviral vector specific for Her-2.