title: A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.
abstract: Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620 ± 49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.
authors.affiliation: College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China.
authors.forename: Enqi
authors.initials: E
authors.lastname: Du
authors.affiliation: College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
authors.affiliation: College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Tikoo
authors.affiliation: College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China.
authors.forename: Zengqi
authors.initials: Z
authors.lastname: Yang
mesh_terms: D025662:Adenoviruses, Porcine / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism*; D016679:Genome, Viral; D016254:Mutagenesis, Insertional / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D034421:Sus scrofa; D013698:Templates, Genetic; D014161:Transduction, Genetic; D019076:Transgenes; D019065:Virus Assembly