Construction of lentiviral vectors containing rat GDNF and TH bi-gene and expression by improved Tet-On system

Aim To construct the lentiviral vector car-rying rat glial cell line-derived neurotrophic factor( GDNF) and tyrosine hydroxylase( TH) on the basis of improved Tet-On system and to investigate the regulation expression of GDNF and TH by tetracycline antibiotics. Methods The rat GDNF and TH genes were obtained with Polymerase Chain Reaction ( PCR) . The GDNF and TH genes were cloned into the lentiviral transfer vector which could be regulated by the improved Tet-On system,in which tetracycline induced transactivator was rtTA2s-M2 and tracycline responsive element was mouse albumin gene promoter( Palb) . Lentiviral supernatants were packaged through lentivirus four plasmids co-transfected into human embryonic kidney cell line-293T by Lipofectamine 2000. Then the virus titer was examined by real-time fluorescence quantitative PCR ( real-time qPCR) . The 293T cells were infected by obtained lentiviral-GDNF-TH and rtTA2S-M2 virus in the identical multiplicity of infection( MOI) . The regulation expression of GDNF and TH mRNA and protein were examined by real-time qPCR and Western blot in different concentrations of doxycycline. Results Tests showed that the recombinant lentiviral transfer vector plasmids were constructed correctly: the titer of lentiviral vector particles was 2. 1 × 1011 TU·L -1; the realtime qPCR and Western blot showed increased expression of mRNA and clear protein bands of GDNF and TH in the Dox-positive group respectively. But they couldn't be detected in the Dox-negative group. Conclusions The present results suggest that lentiviral vector regulated by the improved Tet-On system carrying GDNF and TH genes has been constructed successfully. The expression of GDNF and TH is regulated by tetracycline antibiotics and it shows no basal expression.