McVey Duncan, Zuber Mohammed, Ettyreddy Damodar, Brough Douglas E, Kovesdi Imre
GenVec, Inc., Gaithersburg, Maryland 20878, USA.
J Virol. 2002 Apr;76(8):3670-7. doi: 10.1128/jvi.76.8.3670-3677.2002.
Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.
腺病毒载体的持续改进需要对新型基因组结构进行研究。由于腺病毒可通过用从质粒DNA分离的病毒基因组转染包装细胞系直接产生，因此有可能将基因组构建与病毒生产分开。通过这种方式，无法产生病毒与无法生成所需基因组无关。我们开发了一种基于λ噬菌体的新型系统，该系统允许通过大肠杆菌中的双同源重组快速修饰病毒基因组。重组反应和新生成的基因组可存在于重组缺陷型细菌宿主中，以提高质粒稳定性。此外，该过程不依赖于任何限制性内切酶。该策略依赖于四个主要步骤：（i）腺病毒黏粒与供体质粒之间的同源重组（供体质粒携带所需的修饰和侧翼同源区域，以指导其重组到病毒基因组中）；（ii）在有生产性的λ噬菌体感染期间对重组腺病毒黏粒进行体内包装；（iii）用产生的裂解物转导重组缺陷型大肠杆菌λ溶原菌（溶原菌抑制用于包装重组腺病毒黏粒的辅助噬菌体对宿主细菌进行生产性感染和破坏）；（iv）有效地选择所需的双重组黏粒。每次反应可回收约10,000个双重组黏粒，基本上所有这些都是正确的双重组分子。该系统用于快速高效地产生E1/E3和E1/E3/E4区域缺失的腺病毒基因组。该技术的基础允许对病毒基因组的任何区域进行轻松修饰，以研究新型结构。
