Protocol abstract
Nature Protocols 1, - 701 - 706 (2006)
Published online: 6 July 2006 | doi:10.1038/nprot.2006.93
Subject Categories: Genetic modification | Immunological techniques | Genomics and proteomics | Microbiology and virology | Nucleic acid based molecular biology | Synthetic chemistry
Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles
James T Koerber1, Narendra Maheshri2, Brian K Kaspar3 & David V Schaffer1
Abstract
Rational design of improved gene delivery vehicles is a challenging and potentially time-consuming process. As an alternative approach, directed evolution can provide a rapid and efficient means for identifying novel proteins with improved function. Here we describe a methodology for generating very large, random adeno-associated viral (AAV) libraries that can be selected for a desired function. First, the AAV2 cap gene is amplified in an error-prone PCR reaction and further diversified through a staggered extension process. The resulting PCR product is then cloned into pSub2 to generate a diverse (>106) AAV2 plasmid library. Finally, the AAV2 plasmid library is used to package a diverse pool of mutant AAV2 virions, such that particles are composed of a mutant AAV genome surrounded by the capsid proteins encoded in that genome, which can be used for functional screening and evolution. This procedure can be performed in approximately 2 weeks.
- Department of Chemical Engineering and the Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720-1462, USA.
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
- Department of Gene Therapy and Division of Molecular Medicine, Columbus Children's Research Institute and Ohio State University, Columbus, Ohio 43205, USA.
Correspondence to: David V Schaffer1 e-mail: schaffer@berkeley.edu
nature-products
A-Z product listing
- AAV(ATCC)
- Agarose(Cambrex)
- ClaI(NEB)
- dNTPs(Invitrogen)
- ElectroMAX DH10B cells(Invitrogen)
- Escherichia coli (Invitrogen)
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