Tsunoda H, Hayakawa T, Sakuragawa N, Koyama H
Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Yokohama, 244-0813, Japan.
Virology. 2000 Mar 15;268(2):391-401. doi: 10.1006/viro.1999.0122.
Adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on human chromosome 19q13.3-qter. Similarly, there is accumulating evidence that this site-specific integration occurs by transfection of AAV-based plasmid vectors. In order to further define the process of plasmid integration events, we constructed some AAV plasmids, introduced them into HeLa cells by lipofection, and isolated chromosomal integrants. One of such plasmids, pTH-5, contained the rep and neomycin-resistant (neo(r)) genes flanked by the 5'- and 3'-inverted terminal repeats of AAV and the hygromycin-resistant (hyg(r)) gene located in the plasmid backbone. Southern blot analysis revealed that among 36 G418-resistant (G418(r)) clones isolated, 22 (61%) showed site-specific integration into AAVS1. Further structural and functional analyses on the expression of the hyg(r) gene in the site-specific clones and the LacZ gene in clones generated with plasmid pTH-2 indicated that, together with the AAV sequence, the plasmid backbone was integrated into the AAVS1 site and thus the neo(r) and hyg(r) genes remained linked at high frequencies in the targeted integrants compared with random integrants. Sequence analysis of integration junctions between pTH-5 and AAVS1 revealed that the junctions occurred in the p5 promoter region of the plasmid while mainly in the partial cDNA coding region of the AAVS1 site. We also found that plasmid pTH-1 linearized in the backbone before lipofection gave a significantly lower frequency of site-specific integration (26%) than the circular form (60%). This finding may support the involvement of the double-stranded, circular form of infected AAV in the integration process. Our results may help to understand the process and mechanism of site-specific integration of lipofected AAV plasmid vectors.
腺相关病毒（AAV）特异性整合到人19号染色体q13.3 - qter上的一个位点（AAVS1）。同样，越来越多的证据表明，这种位点特异性整合是通过转染基于AAV的质粒载体发生的。为了进一步明确质粒整合事件的过程，我们构建了一些AAV质粒，通过脂质体转染将它们导入HeLa细胞，并分离出染色体整合体。其中一个这样的质粒pTH - 5，包含位于AAV的5'和3'反向末端重复序列两侧的rep和新霉素抗性（neo(r)）基因，以及位于质粒骨架中的潮霉素抗性（hyg(r)）基因。Southern印迹分析显示，在分离出的36个G418抗性（G418(r)）克隆中，有22个（61%）显示出对AAVS1的位点特异性整合。对位点特异性克隆中hyg(r)基因和用质粒pTH - 2产生的克隆中LacZ基因表达的进一步结构和功能分析表明，与AAV序列一起，质粒骨架被整合到AAVS1位点，因此与随机整合体相比，neo(r)和hyg(r)基因在靶向整合体中以高频率保持连锁。pTH - 5与AAVS1之间整合连接点的序列分析表明，连接点发生在质粒的p5启动子区域，而主要在AAVS1位点的部分cDNA编码区域。我们还发现，脂质体转染前在骨架中线性化的质粒pTH - 1产生的位点特异性整合频率（26%）明显低于环状形式（60%）。这一发现可能支持感染的AAV双链环状形式参与整合过程。我们的结果可能有助于理解脂质体转染的AAV质粒载体位点特异性整合的过程和机制。
