Enqi Du1, Suresh K Tikoo. 1. Vaccine and Infectious Disease Organization, University of Saskatchewan Saskatoon, Saskatchewan, Canada.
Abstract
BACKGROUND: The rigorous evaluation of recombinant bovine adenovirus (BAdV)-3 as a gene delivery vector requires quick and efficient method of isolating recombinants. This requires both a suitable cell line and an efficient method of rescuing recombinant BAdV-3. To facilitate rapid isolation of recombinant BAdV-3, we have developed an efficient system for generating recombinants using newly identified nonbovine cell line permissive for replication of BAdV-3. METHODS: Nonbovine cotton rat lung (CRL) cells in comparison to Madin-Darby bovine kidney cells and VIDO R2 cells were analyzed for the production of progeny virus and DNA transfection efficiency. In addition, lentiviral expression system was used to generate stable nonbovine CRL cell line expressing endonuclease I-SceI as examined by western blotting. Transfection of this cell line with circular or linear plasmid containing full-length BAdV-3 genome was used to generate recombinant BAdV-3. RESULTS: We demonstrate that nonbovine CRL cells are permissive for replication of BAdV-3 and can be efficiently transfected with plasmid DNA. Second, we constructed CRL cell line (VIDO DT1) expressing an intron-encoding endonuclease I-SceI. Finally, we demonstrate that transfection of VIDO DT1 cells with a circular plasmid containing recombinant BAdV-3 genome flanked by I-SceI recognition sites can efficiently rescue recombinant virus. CONCLUSIONS: The use of circular molecular clones together with I-SceI endonuclease expressing, BAdV-3 permissive CRL cell line not only increased the viral genome transfection efficiency, but also reduced the viral rescue time and amount of DNA required for rescuing recombinant BAdV-3s.
BACKGROUND: The rigorous evaluation of recombinant bovine adenovirus (BAdV)-3 as a gene delivery vector requires quick and efficient method of isolating recombinants. This requires both a suitable cell line and an efficient method of rescuing recombinant BAdV-3. To facilitate rapid isolation of recombinant BAdV-3, we have developed an efficient system for generating recombinants using newly identified nonbovine cell line permissive for replication of BAdV-3. METHODS: Nonbovine cotton rat lung (CRL) cells in comparison to Madin-Darby bovine kidney cells and VIDO R2 cells were analyzed for the production of progeny virus and DNA transfection efficiency. In addition, lentiviral expression system was used to generate stable nonbovine CRL cell line expressing endonuclease I-SceI as examined by western blotting. Transfection of this cell line with circular or linear plasmid containing full-length BAdV-3 genome was used to generate recombinant BAdV-3. RESULTS: We demonstrate that nonbovine CRL cells are permissive for replication of BAdV-3 and can be efficiently transfected with plasmid DNA. Second, we constructed CRL cell line (VIDO DT1) expressing an intron-encoding endonuclease I-SceI. Finally, we demonstrate that transfection of VIDO DT1 cells with a circular plasmid containing recombinant BAdV-3 genome flanked by I-SceI recognition sites can efficiently rescue recombinant virus. CONCLUSIONS: The use of circular molecular clones together with I-SceI endonuclease expressing, BAdV-3 permissive CRL cell line not only increased the viral genome transfection efficiency, but also reduced the viral rescue time and amount of DNA required for rescuing recombinant BAdV-3s.
Authors: Jin Huk Choi; Stephen C Schafer; Lihong Zhang; Terry Juelich; Alexander N Freiberg; Maria A Croyle Journal: Mol Pharm Date: 2013-08-19 Impact factor: 4.939