Annexin A1 (ANXA1) protein is involved in the regulation of cell apoptosis.OBJECTIVE:
To construct a short hairpin RNA (shRNA) lentiviral vector targeting rabbit ANXA1 gene and to detect its efficiency of gene silence in osteoblasts.METHODS:
Targeting rabbit ANXA1 gene sequences, a pair of complementary small hairpin RNA (shRNA) oligonucleotides were designed, synthesized, annealed and cloned into pGLV/H1/GFP plasmid digested by BamHI and EcoRI to construct a vector named pGLV-shANXA1. The recombinant plasmid was identified by PCR and DNA sequencing. 293FT cells were co-transfected with pGLV-shANXA1, pGLV/helper-1,pGLV/helper-2 and pGLV/helper-3 to package into lentivirus particles named LV-shANXA1.The titer of virus was tested according to the expression level of GFP. The lentivirus particles were then transmitted into rabbit osteoblasts and its infection efficiency was assessed by flow cytometry.RESULTS AND CONCLUSION:
The PCR identification and DNA sequencing showed that the fragment and nucleotides were in accordance with the target sequence and the shRNA sequence was successfully inserted into the pGLV/H1/GFP vector. The titer of concentrated LV-shANXA1 was 3.8×108 TU/L. The infection efficiency of lentiviral particles was 80% at multiplicity of infection (MOI) of 50 and 95% at MOI of 100 in osteoblasts after puromycin selection. Real-time PCR and Western blot results showed that the expression of ANXA1 in LV-shANXA1-901 infected osteoblasts were lower than that in the blank control, negative control, and other LV-shANXA1 infected cells (P < 0.05), it could achieve 71.2% interference efficiency at MOI of 50. The results demonstrate a lentiviral shRNA expression vector targeting the ANXA1 gene is successfully constructed and it can knockdown the expression of ANXA1 in osteoblasts.RESULTS AND CONCLUSION:
Liang BW, Pan XY, Zhao JM, Yin GQ, Hu F. Construction of short hairpin RNA lentiviral vector targeting rabbit Annexin A1 gene and identification of RNA interference efficiency. Zhongguo Zuzhi Gongcheng Yanjiu. 2012;16(11): 1954-1958.