OBJECTIVE: Glutamic acid decarboxylase 2 (GAD65) is a gamma-aminobutyric acid (GABA) synthetase. This study aimed to construct a recombinant lentivirus-rGAD65 (rLV-rGAD65) vector containing the cDNA of rat GAD65 (rGAD65) and assess its functional activity in vitro and in vivo. METHODS: cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector, forming the rLV-GFP-rGAD65 plasmid. The recombinant lentivirus particles (rLV-rGAD65) were packaged by the LV Helper-Free System and the titer was measured. Primary rat lung fibroblasts were transfected with rLV-rGAD65. The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph (HPLC). In vivo, rLV-rGAD65 was injected into the subthalamic nucleus (STN) of Sprague-Dawley rats using stereotaxic methods, and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot, while the GABA concentration in the substantia nigra pars reticulata (SNr) was assayed by HPLC. RESULTS: The sequence of rGAD65 cDNA was in accord with that in GenBank. The amino-acid sequence of rGAD65 had no mutations and the titer of rLV-rGAD65 reached 6.8 × 10⁸/mL. The efficiency of infection of fibroblasts was 80%, and the concentration of GABA in the medium was (48.14 +/- 9.35) nmol/L. In vivo, rGAD65 expression was detected in the STN, and the concentration of GABA in the SNr increased from (5.95 +/- 1.09) to (12.44 +/- 3.79) nmol/g tissue. CONCLUSION: The recombinant LV-GFP-rGAD65 vector was successfully constructed. rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid. In vivo, the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.
OBJECTIVE:Glutamic acid decarboxylase 2 (GAD65) is a gamma-aminobutyric acid (GABA) synthetase. This study aimed to construct a recombinant lentivirus-rGAD65 (rLV-rGAD65) vector containing the cDNA of ratGAD65 (rGAD65) and assess its functional activity in vitro and in vivo. METHODS: cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector, forming the rLV-GFP-rGAD65 plasmid. The recombinant lentivirus particles (rLV-rGAD65) were packaged by the LV Helper-Free System and the titer was measured. Primary rat lung fibroblasts were transfected with rLV-rGAD65. The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph (HPLC). In vivo, rLV-rGAD65 was injected into the subthalamic nucleus (STN) of Sprague-Dawley rats using stereotaxic methods, and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot, while the GABA concentration in the substantia nigra pars reticulata (SNr) was assayed by HPLC. RESULTS: The sequence of rGAD65 cDNA was in accord with that in GenBank. The amino-acid sequence of rGAD65 had no mutations and the titer of rLV-rGAD65 reached 6.8 × 10⁸/mL. The efficiency of infection of fibroblasts was 80%, and the concentration of GABA in the medium was (48.14 +/- 9.35) nmol/L. In vivo, rGAD65 expression was detected in the STN, and the concentration of GABA in the SNr increased from (5.95 +/- 1.09) to (12.44 +/- 3.79) nmol/g tissue. CONCLUSION: The recombinant LV-GFP-rGAD65 vector was successfully constructed. rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid. In vivo, the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.
Authors: Deniz Kirik; Biljana Georgievska; Corinna Burger; Christian Winkler; Nicholas Muzyczka; Ronald J Mandel; Anders Bjorklund Journal: Proc Natl Acad Sci U S A Date: 2002-03-26 Impact factor: 11.205
Authors: Jia Luo; Michael G Kaplitt; Helen L Fitzsimons; David S Zuzga; Yuhong Liu; Michael L Oshinsky; Matthew J During Journal: Science Date: 2002-10-11 Impact factor: 47.728