Construction and screening of lentiviral vector of RNA interference of mouse XBP1 gene

Objective: To construct lentiviral vector of RNA interference(RNAi)of mouse X-box binding protein 1 (XBP1) gene, and screen the effective sequence of siRNA targeting XBP1 gene. Methods: According to the GenBank information of mouse XBP1 gene, four interfering sequences and a negative sequence were designed. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was synthesized, and then were formed into double-stranded DNA by annealing. The obtained products were cloned into the pGCL-GFP vector digested by the restriction enzyme of AgeⅠand EcoRⅠ to construct lentiviral vector which expressed short hairpin RNA (shRNA),and it was identified by PCR and DNA sequencing. The successful construct vector was packaged by packaging plasmid mix. The titer of virus was tested. NIH3T3 cells were infected by lentivirus, and the interfering efficiency was determined by Real-time PCR. Results: PCR identification and DNA sequencing demonstrated that insertion of oligonucleotide was correct. The titer of virus tested according to the expression level of GFP was 1 ×108 TU/ml. Real-time PCR anaylsis confirmed XBP1-siRNA-3 had the highest interfering efficiency, and the interfering efficiency of lentivirus was more 95% . Conclusion: The lentivirus RNAi vector of mouse XBP1 was constructed and screened successfully, which lays a foundation for further study of the role of XBP1 in the regulation of macrophage immune function.