Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles
Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles
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DOI：
10.1038/nprot.2006.93
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Schaffer, David V.
中科院分区：
文献类型：
--
作者：
Koerber, James T.;Maheshri, Narendra;Schaffer, David V.
合理设计改进的基因输送工具是一个具有挑战性且可能非常耗时的过程。作为一种替代方法，定向进化可以提供一种快速有效的手段来鉴定具有更高功能的新蛋白质。在这里，我们描述了一种产生非常大的随机腺相关病毒(AAV)文库的方法，该文库可以被选择用于所需的功能。首先，AAV2帽基因在容易出错的PCR反应中被扩增，并通过交错延伸过程进一步多样化。然后将所得的聚合酶链式反应产物克隆到pSub2中，以产生一个不同的(>；10(6))AAV2质粒库。最后，AAV2质粒库被用来包装不同的突变型AAV2病毒粒子池，使得颗粒由突变的AAV2病毒基因组组成，该基因组被编码在该基因组中的衣壳蛋白包围，可用于功能筛选和进化。这一过程可以在大约2周内完成。
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.