Hybrid yeast-bacteria cloning system used to capture and modify adenoviral and nonviral genomes
Hybrid yeast-bacteria cloning system used to capture and modify adenoviral and nonviral genomes
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DOI：
10.1089/104303403321208934
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发表时间：
2003-03-01
影响因子：
4.2
通讯作者：
Mendez, MJ
中科院分区：
文献类型：
--
作者：
Hokanson, CA;Dora, E;Mendez, MJ
腺病毒载体在体外和体内广泛用于表达转基因。腺病毒载体产生的一个主要障碍是对大的(35kb)腺病毒基因组的操作。我们开发了一种酵母-细菌混合克隆系统来创建新腺病毒载体。腺病毒5型(Ad5)基因组被克隆到同时含有酵母和细菌元件的穿梭载体中进行复制，因此既具有酵母人工质粒(YAP)的功能，又具有质粒人工染色体(PAC)的功能。任何序列都可以通过酵母中高效的同源重组引入到腺病毒基因组的任何区域，然后这些重组体在细菌中被快速扩增。腺病毒载体是通过将PAC导入适当的互补哺乳动物细胞而产生的，而不需要进行菌斑纯化。构建的载体带有E1、E3和/或E4区域的缺失。我们已经产生了100多个带有许多不同转基因和调控元件的载体。此外，利用YAP/PAC载体捕获了包含人凝血因子IX基因的DNA片段，证明了该系统在克隆和分析基因组DNA方面的有效性。这一新的克隆策略允许快速和多功能地构建用于基因表达和基因治疗应用的腺病毒载体。
Adenoviral vectors are widely used to express transgenes in vitro and in vivo. A major obstacle to the generation of adenoviral vectors is the manipulation of the large (35 kb) adenoviral genome. We developed a hybrid yeast-bacteria cloning system for the creation of novel adenoviral vectors. The adenovirus 5 (Ad5) genome was cloned into a shuttle vector that contains both yeast and bacterial elements for replication and therefore functions as both a yeast artificial plasmid ( YAP) and as a plasmid artificial chromosome (PAC). Any sequence can be introduced into any region of the adenoviral genome via the highly efficient homologous recombination in yeast and then these recombinants are rapidly amplified in bacteria. Adenoviral vectors are generated by introduction of the PAC into the appropriate complementing mammalian cell without the need for plaque purification. Vectors were constructed with deletions in the E1, E3, and/or E4 regions. We have generated more than 100 vectors with a number of different transgenes and regulatory elements. In addition, the YAP/PAC vector was used to capture a DNA fragment encompassing the human factor IX gene, demonstrating the utility of this system to clone and analyze genomic DNA. This novel cloning strategy allows the rapid and versatile construction of adenoviral vectors for gene expression and gene therapy applications.