Department of Vaccinology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Institute of Virology, Hannover Medical School, Hannover, Germany.
Methods Mol Biol. 2021;2244:133-158. doi: 10.1007/978-1-0716-1111-1_8.
To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they are studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of these loss-of-function viral mutants to the wild-type virus allow for the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 20 years. The cloning of CMV genomes into Escherichia coli as bacterial artificial chromosomes (BAC) allows for not only quick and efficient deletion of viral genomic regions, individual genes, or single-nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain-of-function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined parental viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, providing selective growth advantage to the recombined genomes and thus clonal selection of viruses with even extremely poor fitness. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.
为了充分了解巨细胞病毒 (CMV) 基因的功能，必须在感染的背景下研究它们。因此，靶向删除单个病毒基因，并将这些功能丧失病毒突变体与野生型病毒进行比较，可以确定特定基因在病毒复制周期中的相关性和作用。靶向 CMV 诱变在过去 20 年中取得了巨大进展。将 CMV 基因组克隆到大肠杆菌中的细菌人工染色体 (BAC) 不仅允许快速有效地删除病毒基因组区域、单个基因或病毒基因组中的单核苷酸交换，还允许插入异源遗传序列以进行功能获得方法。这种策略的概念优势在于它克服了细胞培养系统中重组技术的限制。即，感染细胞中的重组仅发生在少数几个克隆中，如果目标基因与病毒复制相关并且不能与未重组的亲本病毒竞争生长，则无法选择这些基因。另一方面，BAC 诱变使能够在大肠杆菌中进行抗生素抗性的选择，为重组基因组提供选择性生长优势，从而对即使适应性非常差的病毒进行克隆选择。在这里，我们描述了生成 CMV BAC、靶向 BAC 克隆突变和用 CMV BAC DNA 转染人细胞以重新构建病毒感染过程的方法。
