Sandalon Z, Gnatenko D V, Bahou W F, Hearing P
Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-5222, USA.
J Virol. 2000 Nov;74(22):10381-9. doi: 10.1128/jvi.74.22.10381-10389.2000.
Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for transgene expression. We have exploited the unique features of the adeno-associated virus (AAV) terminal repeats within the context of an adenovirus-adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recombination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mAd DNA exists as monomer and dimer forms within the recombinant viral capsid. Formation of recombinant mAd was significantly increased using an AAV Rep78- or Rep68-expressing cell line through Rep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able to transfer functional gene to A549 and HeLa cells. This approach is rapid and efficient, thereby providing a simplified methodology for generating mAd with functional transducing capabilities.
删除所有病毒编码区的微型腺病毒（mAd）代表了一种新兴的转基因表达方法。我们利用腺病毒-腺相关杂交病毒（Ad/AAV）背景下腺相关病毒（AAV）末端重复序列的独特特征，作为快速高效生成mAd的策略。通过重组在293细胞中从亲本Ad/AAV杂交载体切除并生成mAd，但无需选择mAd生产。对从293细胞中分离出的mAd的分析表明，mAd DNA以单体和二聚体形式存在于重组病毒衣壳内。通过利用Ad/AAV杂交病毒基因组中存在的AAV末端重复序列，通过Rep介导的切除，使用表达AAV Rep78或Rep68的细胞系，重组mAd的形成显著增加。mAd病毒具有感染性，能够将功能性基因转移到A549和HeLa细胞中。这种方法快速高效，从而为生成具有功能性转导能力的mAd提供了一种简化的方法。
