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Biotechniques. 2014 May 1;56(5):263-8. doi: 10.2144/000114169. eCollection 2014 May.

Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning.

Author information

1
Department of Biomedicine, Faculty of Heath, Aarhus University, Aarhus, Denmark.
2
Department of Biomedicine, Faculty of Heath, Aarhus University, Aarhus, Denmark; Department of Clinical Medicine, Department of Cardiological Medicine B, Aarhus University, Aarhus, Denmark.

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.

KEYWORDS:

Golden Gate cloning; gene targeting; p53; rAAV; tau

PMID:
24806227
DOI:
10.2144/000114169
[Indexed for MEDLINE]
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