Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS
Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS
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DOI：
10.1016/j.jviromet.2006.03.014
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发表时间：
2006-08-01
影响因子：
3.1
通讯作者：
Leib, David A.
中科院分区：
文献类型：
--
作者：
Gierasch, William W.;Zimmerman, David L.;Leib, David A.
构建了含有HSV-1株17和KOS全长感染性DNA的细菌人工染色体（BACs）。为了在不改变病毒基因的情况下生成BAC，将BAC选择和繁殖所需的序列置于U(L)37和U(L)38基因之间，并在两侧放置LoxP位点。通过在体外和体内研究这些HSV-1 BAC构建物的多种特性，在细菌中繁殖，在真核细胞中从HSV-BAC DNA中重构病毒，以及通过cre重组酶介导的BAC主干切除，对该系统进行了测试。基于小鼠胚胎成纤维细胞的体外生长和小鼠角膜和三叉神经节的体内生长，菌株KOS bac衍生病毒的表现与野生型相似。然而，观察到神经毒性的微小变化。与野生型相比，菌株17 bac衍生病毒的生长和毒力略有下降。在KOS毒株和17种bac衍生病毒的潜伏期再激活中观察到适度的差异。此外，通过在大肠杆菌中进行等位基因交换对BACs进行诱变，进一步验证了该系统。这些BACs适合快速生成重组病毒用于致病机制和其他研究，但与所有诱变系统一样，必须注意其构建和修复。(c) 2006 Elsevier B.V.版权所有
Bacterial artificial chromosomes (BACs) were constructed containing full-length, infectious DNA of HSV-1 strains 17 and KOS. To generate BACs without altering viral genes, sequences required for selection and propagation of the BAC were placed between the U(L)37 and U(L)38 genes, and flanked by LoxP sites. The system was tested by studying multiple properties of these HSV-1 BAC constructs in vitro and in vivo following propagation in bacteria, virus reconstitution from HSV-BAC DNA in eukaryotic cells, and Cre-recombinase-mediated excision of the BAC backbone. Based on in vitro growth in mouse embryo fibroblasts and in vivo growth in mouse corneas and trigeminal ganglia, the strain KOS BAC-derived virus behaved similarly to wild-type. Small changes in neurovirulence were, however, observed. The strain 17 BAC-derived virus exhibited modest decreases in growth and virulence compared to wild-type. Modest differences were observed in reactivation from latency with both strain KOS and 17 BAC-derived viruses. In addition, the system was further validated by performing mutagenesis of the BACs by allelic exchange in E. coli. These BACs are suitable for the rapid generation of recombinant viruses for pathogenesis and other studies, but as with all mutagenesis systems, care must be taken in their construction and repair. (c) 2006 Elsevier B.V. All rights reserved.