Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles

被引:60
作者
Koerber, James T.
Maheshri, Narendra
Kaspar, Brian K.
Schaffer, David V. [1 ]
机构
[1] Univ Calif Berkeley, Helen Wills Neurosci Inst, Dept Chem Engn, Berkeley, CA 94720 USA
[2] MIT, Dept Chem Engn, Cambridge, MA 02139 USA
[3] Ohio State Univ, Columbus Childrens Res Inst, Dept Gene Therapy, Div Mol Med, Columbus, OH 43205 USA
关键词
D O I
10.1038/nprot.2006.93
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 (> 10(6)) 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.
引用
收藏
页码:701 / 706
页数:6
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