He T C, Zhou S, da Costa L T, Yu J, Kinzler K W, Vogelstein B
The Howard Hughes Medical Institute, 424 North Bond Street, Baltimore, MD 21231, USA.
Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2509-14. doi: 10.1073/pnas.95.5.2509.
Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.
重组腺病毒为基因表达研究和治疗应用提供了一个多功能系统。我们在此报道一种简化此类病毒产生和生产的策略。使用细菌中的同源重组而非真核细胞中的同源重组，通过最少的酶促操作产生重组腺病毒质粒。将此类质粒转染到哺乳动物包装细胞系后，借助由整合到病毒骨架中的基因编码的绿色荧光蛋白，可方便地跟踪病毒生产情况。无需空斑纯化即可从此过程中获得均匀的病毒。该系统应能加快为各种目的产生和测试重组腺病毒的过程。
