Kojima H, Ohishi N, Yagi K
Gifu International Institute of Biotechnology, Japan.
Biochem Biophys Res Commun. 1998 May 29;246(3):868-72. doi: 10.1006/bbrc.1998.8706.
To perform the complex manipulation of the adenoviral genome for the construction of recombinant adenovirus vectors, we developed a cosmid vector (pacad1A) from which an infectious E1 and E3-deleted adenoviral genome can be released with PacI digestion. The cosmid vector, pacad1A, has unique restriction enzyme sites that are created for the insertion of foreign genes into the deleted E1 or E3 region of the adenoviral genome. To demonstrate the feasibility of the construction of adenovirus vectors with our developed vector, we showed that a recombinant adenovirus bearing a self-contained tetracycline-regulated expression system could be generated by transfection of cells with an infectious adenoviral genome that was released from pacad1A-derived plasmid DNA. The recombinant adenovirus vector was obtained easily by this method, and the expression of a transgene was proved to be regulated with tetracycline in CHO-K1 cells.
为了对腺病毒基因组进行复杂操作以构建重组腺病毒载体，我们开发了一种黏粒载体（pacad1A），通过PacI酶切可从中释放出具有感染性的E1和E3缺失的腺病毒基因组。黏粒载体pacad1A具有独特的限制性酶切位点，这些位点是为了将外源基因插入腺病毒基因组的缺失E1或E3区域而创建的。为了证明用我们开发的载体构建腺病毒载体的可行性，我们表明，通过用从pacad1A衍生的质粒DNA释放的具有感染性的腺病毒基因组转染细胞，可以产生携带自包含四环素调控表达系统的重组腺病毒。通过这种方法很容易获得重组腺病毒载体，并且证明在CHO-K1细胞中，转基因的表达受四环素调控。
