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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cinnamates / pharmacology
  • Cloning, Molecular / methods*
  • Dependovirus / genetics*
  • Drug Resistance, Microbial / genetics
  • Gene Knockout Techniques
  • Gene Targeting / methods*
  • Genetic Engineering / methods
  • Genetic Vectors*
  • Humans
  • Hygromycin B / analogs & derivatives
  • Hygromycin B / pharmacology
  • Neomycin / pharmacology
  • Reproducibility of Results
  • tau Proteins / genetics

Substances

  • Cinnamates
  • Mapt protein, mouse
  • tau Proteins
  • Hygromycin B
  • hygromycin A
  • Neomycin