Nucleic Acids Research, 2003, Vol. 31, No. 14 e76
© 2003 Oxford University Press
Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
*To whom correspondence should be addressed. Tel: +81 3 54495627; Fax: +81 3 5449 5432; Email: isaito{at}ims.u-tokyo.ac.jp
Present addresses:
Aya Okuda, Scientific Information Services Group, Marketing Department, Research Products Division, Daiichi Pure Chemicals Co. Ltd, 2-8-3 Higashinihonbashi, Chuo-ku, Tokyo 103-0004, Japan
Hiromi Sato, Quality Control Section, Production Department, Pharmaceutical Division, Kirin Brewery Co. Ltd, 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan
Naoto Tachikawa, Zeria Pharmaceutical Co. Ltd, 10-11 Nihonbashi, Kobuna-cho, Chuo-ku, Tokyo 103-8351, Japan
Miho Terashima, Gene Engineering Division, BioResource Center RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
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.
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