Department of Comparative Pathobiology, School of Veterinary Medicine, and Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
Virus Res. 2011 Nov;161(2):181-7. doi: 10.1016/j.virusres.2011.08.002. Epub 2011 Aug 16.
The state of vector genome in transduced cells influences the duration of transgene expression and can be a safety concern if it gets integrated randomly into the host genome. Although human adenovirus (Ad) serotype 5 (HAd5) mainly persists in a linear episomal form, information regarding the state of bovine Ad serotype 3 (BAd3) and porcine Ad serotype 3 (PAd3) vector genomes in human and nonhuman cells is currently unknown. To address this issue, MDA-MB-231 (human), MDBK (bovine), PK-15 (porcine), MT1A2 (mouse) and NIH-3T3 (mouse) cell lines were infected with replication-defective BAd3, PAd3 or HAd5 vectors carrying the green fluorescent protein (GFP) gene. The persistence and the state of vector genome were assessed by quantitative real-time PCR and Southern blot hybridization, respectively. Levels of transgene and Ad gene expressions were quantified using real-time RT-PCR. Persistence of BAd3 or PAd3 vectors was comparable to that of HAd5 vector. Only the linear episomal form of the vector genome was observed with each vector. In addition, expression levels of transgene as well as viral genes by all three vectors were comparable and correlated with their transduction levels in each cell type. These results indicate comparable biologic behavior of BAd3, PAd3 and HAd5 vectors in cell culture.
转导细胞中的载体基因组状态会影响转基因的表达时间，如果它随机整合到宿主基因组中，可能会成为一个安全隐患。虽然人类腺病毒（Ad）血清型 5（HAd5）主要以线性附加体形式存在，但目前尚不清楚牛腺病毒（BAd3）和猪腺病毒（PAd3）载体基因组在人和非人类细胞中的状态。为了解决这个问题，用携带绿色荧光蛋白（GFP）基因的复制缺陷型 BAd3、PAd3 或 HAd5 载体感染 MDA-MB-231（人）、MDBK（牛）、PK-15（猪）、MT1A2（鼠）和 NIH-3T3（鼠）细胞系。分别通过定量实时 PCR 和 Southern 印迹杂交评估载体基因组的持续性和状态。使用实时 RT-PCR 定量转基因和 Ad 基因的表达。BAd3 或 PAd3 载体的持续性与 HAd5 载体相当。每种载体只观察到线性附加体形式的载体基因组。此外，三种载体的转基因表达水平以及病毒基因的表达水平均相当，并且与它们在每种细胞类型中的转导水平相关。这些结果表明 BAd3、PAd3 和 HAd5 载体在细胞培养中具有相似的生物学行为。
