Construction and identification of recombinant lentiviral vector expressing rat brain derived neurotrophic factor gene and Infection of adipose-derived stem cells

Abstract

Objective To construct a lentiviral brain derived neurotrophic factor (BDNF) expression vector and then infect the adipose derived mesenchymal stem cells (ADSCs) to produce therapeutic seed cells. Methods The BDNF gene was isolated and amplified by polymerase chain reaction (PCR) technique from pEGFP-C-BDNF plasmid which was constructed before. Then the gene was subcloned into the expression plasmid of lentiviral vector, GV287 (containing Flag gene), to generate the lentiviral expression vector, UBI-BDNF. The correct BDNF gene was confirmed by PCR, endoenzyme digestion, sequencing, analysis and comparison. Recombinant lentivirnses containing BDNF gene and nag gene were produced by 293T cells following the co-transfection of UBI-BDNF and packaging plasmids-pHelper1.0 and pHelper2.0. The newly constructed recombinant lentivirus and the titer of virus were confirmed by real-time quantitative polymerase chain reaction (Real-time PCR), and then the ADSCs were transfected with the vectors after titer determination. Stable expression of BDNF in ADSCs was confirmed by immunofluorescence staining, enzyme linked immunosorbent assay (ELISA) and Western blotting. Results The evidence of endonuclease digestion, DNA sequencing and PCR analysis confirmed that BDNF gene was correctly inserted into the lentiviral vector, and the titer of virus was 2.0×109 TU/ml. BDNF could be expressed in the transfected cells confirmed by immunofluorescence staining. By ELISA, the density of BDNF was 891.38-1 196.26 ng/L [(945.33±38.54) ng/L] in the supernatant of BDNF-transfected cells but not detected in the GFP-transfected cells (P<0.05). By Western blotting also confirmed the BDNF expression in BDNF-transfected cells. Conclusion The recombinant lentivirus expressing BDNF was successfully constructed and over-expressed in ADSCs. Key words: Brain derived neurotrophic factor; Lentiviral vector; Gene transfection; Adipose-derived stem cells