Separate control of rep and cap expression using mutant and wild-type LoxP sequences and improved packaging system for adeno-associated virus vector production
Separate control of rep and cap expression using mutant and wild-type LoxP sequences and improved packaging system for adeno-associated virus vector production
复制标题
使用突变型和野生型 LoxP 序列单独控制 rep 和 cap 表达，并改进腺相关病毒载体生产的包装系统
作者：
H. Mizukami;T. Okada;Y. Ogasawara;T. Matsushita;M. Urabe;A. Kume;K. Ozawa
腺相关病毒（AAV）载体是基因转移的实际选择，并且对它们的需求正在增加。为了科普在不久的将来的必要性，我们已经开发了许多方法来建立用于生产AAV载体的包装细胞系。在我们之前的研究中，通过使用Cre-loxP转换系统获得了大Rep蛋白的高度调控表达。因此，在本研究中，为了调节Cap表达，我们通过使用另外一组突变loxP序列开发了Rep和Cap蛋白的诱导表达系统。突变体在loxP的间隔区具有两个碱基改变，并且在Cre存在下与相同的对应物特异性重组。通过使用两个单独的质粒，一个具有突变型loxP序列，另一个具有野生型loxP序列，可以通过Cre重组酶同时诱导两种不同蛋白质的表达。当使用LacZ编码质粒载体作为包装模型时，获得了每10厘米培养皿2.1 × 1010个基因组拷贝的显着包装滴度。这些结果表明，除了Rep之外，控制Cap表达以实现AAV载体的最佳生产速率的重要性。
Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing. To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors. In our previous study, a highly regulated expression of large Rep proteins was obtained by using the Cre-loxP switching system. Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences. The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre. By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase. When the LacZencoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 × 1010 genome copies per 10-cm dish was obtained. These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.
影响因子：
4.2
作者：
Gao, GP;Qu, G;Wilson, JM
通讯作者：
Wilson, JM