Human cytomegalovirus: bacterial artificial chromosome (BAC) cloning and genetic manipulation.
Human cytomegalovirus: bacterial artificial chromosome (BAC) cloning and genetic manipulation.
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人类巨细胞病毒：细菌人工染色体（BAC）克隆和遗传操作。
DOI：
10.1002/9780471729259.mc14e04s24
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发表时间：
2012
影响因子：
--
通讯作者：
Yu,Dong
中科院分区：
文献类型：
--
作者：
Paredes,AnneM;Yu,Dong
由于无法进行有效的病毒遗传分析，对人类巨细胞病毒（HCMV）生物学的认识长期受到阻碍。当多个HCMV菌株的基因组被克隆为感染性细菌人工染色体（BACs）时，这一障碍被克服了。BAC系统利用了e的单拷贝F质粒。大肠杆菌可以稳定地携带大量外源DNA。在该系统中，首先在真核细胞中产生了携带修饰的F质粒的重组HCMV病毒。然后分离重组病毒基因组并回收inE。colias BAC克隆。BAC捕获的病毒基因组可以使用原核遗传学进行操作，并且可以在真核细胞中通过BAC转染重组病毒。BAC反向遗传系统提供了一种可靠和有效的方法来引入基因改变到病毒基因组中。随后分析它们对真核细胞中病毒生物学的影响。Protoc。Microbiol 24:14E.1.1 14 e.1.33应承担的。©2012 by John Wiley & Sons, Inc。
The understanding of human cytomegalovirus (HCMV) biology was long hindered by the inability to perform efficient viral genetic analysis. This hurdle was recently overcome when the genomes of multiple HCMV strains were cloned as infectious bacterial artificial chromosomes (BACs). The BAC system takes advantage of the single‐copy F plasmid ofE. colithat can stably carry large pieces of foreign DNA. In this system, a recombinant HCMV virus carrying a modified F plasmid is first generated in eukaryotic cells. Recombinant viral genomes are then isolated and recovered inE. colias BAC clones. BAC‐captured viral genomes can be manipulated using prokaryotic genetics, and recombinant virus can be reconstituted from BAC transfection in eukaryotic cells. The BAC reverse genetic system provides a reliable and efficient method to introduce genetic alterations into the viral genome inE.coliand subsequently analyze their effects on virus biology in eukaryotic cells.Curr. Protoc. Microbiol. 24:14E.1.1‐14E.1.33. © 2012 by John Wiley & Sons, Inc.