title: Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning.
abstract: The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTau(P301L)-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.
authors.affiliation: Department of Biomedicine, Faculty of Heath, Aarhus University, Aarhus, Denmark.
authors.forename: Yonglun
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Biomedicine, Faculty of Heath, Aarhus University, Aarhus, Denmark.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of Biomedicine, Faculty of Heath, Aarhus University, Aarhus, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.affiliation: Department of Biomedicine, Faculty of Heath, Aarhus University, Aarhus, Denmark; Department of Clinical Medicine, Department of Cardiological Medicine B, Aarhus University, Aarhus, Denmark.
authors.forename: Charlotte Brandt
authors.initials: CB
authors.lastname: Sørensen
mesh_terms: D002934:Cinnamates / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D000229:Dependovirus / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D015203:Reproducibility of Results; D016875:tau Proteins / Q000235:genetics
chemical_list: D002934:Cinnamates; C558298:Mapt protein, mouse; D016875:tau Proteins; D006921:Hygromycin B; C026273:hygromycin A; D009355:Neomycin
keywords: Golden Gate cloning; gene targeting; p53; rAAV; tau