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Journal of Biochemistry Advance Access originally published online on October 27, 2007
Journal of Biochemistry 2008 143(1):87-95; doi:10.1093/jb/mvm198
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© 2007 The Japanese Biochemical Society.

S Phase-preferential Cre-recombination in Mammalian Cells Revealed by HIV-TAT-PTD-mediated Protein Transduction

Mayumi Hashimoto, Makoto Taniguchi, Susumu Yoshino, Shiho Arai and Kenzo Sato*

Division of Molecular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago 683-8503, Japan

*To whom correspondence should be addressed. Tel: +81-859-38-6201, Fax: +81-859-38-6200, E-mail: kensato{at}grape.med.tottori-u.ac.jp

Received October 2, 2007; Accepted October 8, 2007


  Abstract

The Cre recombinase of bacteriophage P1 is a powerful tool for artificial modification of genomic function in mammalian cells. To date, many researchers have studied the enzymatic biochemistry of Cre recombinase in loxP site-specific cleavage and rearrangement, as well as its use in gene technology. However, the intricate mechanisms of Cre-mediated recombination are still poorly understood. For example, more knowledge is needed in order to understand Cre recombinase's dependency on cell cycle, the necessity of other factors for recombination, and the exact nuclear environment that's required at the target locus, in order for recombination to take place in eukaryotic cells. In this study, we showed that P1 Cre-mediated recombination occurred frequently during S-phase of the cell cycle. HeLa cells were synchronized in cell cycle with the thymidine-hydroxyurea block method, and recombinant Cre proteins were fused with HIV-1 TAT protein transduction domains (PTD) in every phase of the cell cycle. Results showed that the transduction of PTD-Cre gave rise to genomic recombination preferentially during the S-phase of cell cycle. These findings will contribute significantly to the development of the Cre/loxP recombination system in vivo.

Key Words: cell cycle, Cre recombinase, GFP, protein transduction, synchronized cell culture

Abbreviations: PTD, protein transduction domain; HIV-1, human immunodeficiency virus-1; β-Gal, β-galactosidase; EGFP, enhanced green fluorescent protein


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