Kondo Saki, Okuda Aya, Sato Hiromi, Tachikawa Naoto, Terashima Miho, Kanegae Yumi, Saito Izumu
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Nucleic Acids Res. 2003 Jul 15;31(14):e76. doi: 10.1093/nar/gng076.
The site-specific recombinase Cre has often been used for on/off regulation of expression of transgenes introduced into the mammalian chromosome. However, this method is only applicable to the regulation of a single gene and cannot be used to simultaneously regulate two genes, because site-specific recombination occurs from the target loxP sequence of one regulating unit to the loxP sequence of any other unit and would eventually disrupt the structure of both regulating units. We previously reported a mutant loxP sequence with a two base substitution called loxP V (previously called loxP 2272), with which wild-type loxP cannot recombine but with which the identical mutant loxP recombines efficiently. In the present study we isolated cell lines bearing two regulating units on a chromosome containing a pair of wild-type loxP sequences or mutant loxP V sequences. After infection with Cre-expressing recombinant adenovirus AxCANCre, expression of a gene in each regulating unit was simultaneously turned on and off. Southern analyses showed that both regulating units were processed simultaneously and independently, even after infection with a limited amount of AxCANCre. The results showed that simultaneous regulation of gene expression on a mammalian chromosome mediated by Cre can be achieved by using mutant loxP V and wild-type loxP. This method may be a useful approach for conditional transgenic/knockout animals and investigation of gene function involving two genes simultaneously. Another possible application is for preparation of a new packaging cell line of viral vectors through simultaneous production of toxic viral genes.
位点特异性重组酶Cre常用于对导入哺乳动物染色体的转基因表达进行开启/关闭调控。然而，该方法仅适用于单个基因的调控，无法用于同时调控两个基因，因为位点特异性重组会从一个调控单元的靶loxP序列发生到任何其他单元的loxP序列，最终会破坏两个调控单元的结构。我们之前报道了一种具有两个碱基替换的突变loxP序列，称为loxP V（之前称为loxP 2272），野生型loxP不能与之重组，但相同的突变loxP能与之高效重组。在本研究中，我们分离出了在含有一对野生型loxP序列或突变loxP V序列的染色体上带有两个调控单元的细胞系。在用表达Cre的重组腺病毒AxCANCre感染后，每个调控单元中基因的表达同时被开启和关闭。Southern分析表明，即使在用有限量的AxCANCre感染后，两个调控单元也是同时且独立地进行处理。结果表明，通过使用突变loxP V和野生型loxP，可以实现由Cre介导的哺乳动物染色体上基因表达的同时调控。该方法对于条件性转基因/基因敲除动物以及同时涉及两个基因的基因功能研究可能是一种有用的方法。另一个可能的应用是通过同时产生有毒病毒基因来制备病毒载体的新包装细胞系。
