A Novel Strategy for Introducing Exogenous Bcl-2 into Neuronal Cells: The Cre/loxP System-Mediated Activation of Bcl-2 for Preventing Programmed Cell Death Using Recombinant Adenoviruses Noboru Sato,* ,1 Siwei Wang,* Ling Li,* Keiko Okabe, ² Mitsuhiro Hashimoto, ‡ Hiroyuki Yaginuma, § Katsuhiko Mikoshiba, ‡,¶, Yasuo Uchiyama,** Taichi Uetsuki, ²² Kazuaki Yoshikawa, ²² Carolanne E. Milligan,* and Ronald W. Oppenheim* *Department of Neurobiology and Anatomy and Neuroscience Program, Wake Forest University School of Medicine, Winston–Salem, North Carolina 27157; † Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305 Japan; ‡ Developmental Neurobiology Laboratory, RIKEN Brain Science Institute, Wako-shi, Saitama 350–01 Japan; § Department of Anatomy, Fukushima Medical College, Fukushima 960–12 Japan; ¶ Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108 Japan;  Calciosignal Net Project, Exploratory Research for Advanced Technology (ERATO), Meguro-ku, Tokyo 153 Japan; **Department of Anatomy 1, Osaka University Medical School, Yamadaoka 2–2, Suita, Osaka 565 Japan; †† Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3–2, Suita, Osaka 565 Japan We have established a novel strategy for introducing exogenous Bcl-2 into neuronal cells that is mediated by Cre/loxP recombination using recombinant adenoviral vec- tors. An on/off-switching cassette for Bcl-2 (CALNLbcl-2) was designed to express Bcl-2 by recombinase Cre- mediated excisional deletion of a spacer DNA flanked by a pair of loxP sites. Exogenous Bcl-2 was clearly induced in PC12 cell lines carrying CALNLbcl-2 after infection with recombinant adenovirus producing recombinase Cre (Ax- CANCre). Dual infection with both AxCANCre and a recom- binant adenovirus bearing CALNLbcl-2 showed efficient delivery of exogenous Bcl-2 into a hybrid motoneuronal cell line and primary chicken spinal motoneurons. The delivery of foreign Bcl-2 promoted survival of motoneu- rons in medium either containing or lacking trophic sup- port. Thus, this strategy for delivery of exogenous Bcl-2 will be useful for studying neuronal death as well as for introducing foreign genes into postmitotic neurons under the control of recombinase Cre. INTRODUCTION A family of genes that share homology with the proto-oncogene bcl-2 play an important role in the regulation of cell death. In the nervous system, Bcl-2 is widely expressed during embryonic development (Merry et al., 1994) and has been shown to protect neurons from various types of cell death, including trophic factor-deprived death in vitro (Garcia et al., 1992; Allsopp et al., 1993) and normal physiological develop- mental neuronal death in vivo (Martinou et al., 1994; Farlie et al., 1995; Bonfanti et al., 1996). Recent in vivo studies using transgenic mice have also shown that Bcl-2 has a neuroprotective effect in various pathologi- cal situations such as axotomy-induced cell death, focal 1 To whom correspondence should be addressed. Fax: (336) 716– 4534. E-mail: nsato@bgsm.edu. MCN Molecular and Cellular Neuroscience 12, 65–78 (1998) Article No. CN980703 65 1044-7431/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.