Neuroscience Letters 399 (2006) 67–70 Bcl-2 expression mediated by Cre/loxP system in olfactory epithelium Kiyoshi Doi a , Ken-ichi Nibu a,∗ , Haruo Okado b , Toshio Terashima c a Department of Otolaryngology-Head and Neck Surgery, Kobe University Graduate School of Medicine, Kusunoki-cho 7-5-1, Chuo-Ku, Kobe 650-0017, Japan b Department of Molecular Physiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan c Department of Anatomy and Developmental Neurobiology, Kobe University Graduate School of Medicine, Kobe, Japan Received 27 November 2005; received in revised form 15 January 2006; accepted 26 January 2006 Abstract To study the Bcl-2 expression mediated by the Cre/loxP recombination system and its effect on prevention of apoptosis in olfactory epithelium. Adenoviral vectors with cassette for Bcl-2 (AxCALNLBcl-2) and Cre recombinase (AxCANCre) were applied to mouse olfactory epithelium by intranasal instillation. The effect of exogenous Bcl-2 expression on prevention of apoptosis of olfactory receptor neurons was investigated using an apoptosis model induced by bulbectomy. The Bcl-2 product was expressed not only in the olfactory receptor neurons but also in the supporting cells. Although statistical analysis did not show significant difference, the number of apoptotic cells in the infected olfactory epithelium on post-bulbectomy day 2 was lower than that of control and the number of survived mature olfactory receptor neurons in the infected olfactory epithelium on post-bulbectomy day 5 was higher than that of control. Although further studies are required for clinical application, the results of our study suggest that this strategy may be able to deliver exogenous Bcl-2 for the treatment of degeneration of olfactory receptor neurons. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Cre/loxP; Olfactory receptor neuron; Adenoviral vector; Bcl-2 The mammalian olfactory receptor neurons (ORNs), which har- bor odorant receptors, are contained in the olfactory epithelium (OE) lining the upper area of the nasal cavity. ORNs have the unusual and characteristic ability to regenerate continuously throughout the lifetime, with dying olfactory neurons being replaced by newly generated ORNs [2]. However, it is thought that olfactory epithelium is degenerated due to toxic environ- mental effects, viruses, toxins, drugs, diseases or smoking [7] as well as ageing [18]. Various growth factors [16] and anti-apoptotic agents [4] have been reported to be essential for olfactory neurogenesis and have been proposed as potential candidate therapeutic drugs for olfactory dysfunction. However, while promising data have been reported based on in vitro experiments, only a few studies using in vivo experiments have been reported [20]. Bcl-2 was originally identified as a protein product of the proto-oncogene Bcl-2 and increased Bcl-2 expression is a com- mon feature of many types of human malignancies, indicating that Bcl-2 plays an important role in carcinogenesis [14]. The ∗ Corresponding author. Tel.: +81 78 382 6020; fax: +81 78 382 6039. E-mail address: nibu@med.kobe-u.ac.jp (K.-i. Nibu). family of this proto-oncogene is vitally involved in the regu- lation of cell death, while Bcl-2 is widely expressed during embryonic development and has been shown to protect neu- rons from various types of cell death [9,15]. Recent in vivo studies using transgenic mice have shown that Bcl-2 also has a neuro-protective effect in various pathological situations [8]. It has also been demonstrated that Bcl-2 promotes regeneration of axons by an action occurring separately from its well-known anti-apoptotic activity [3]. The successful gene delivery to the ORNs in the olfactory system by adenoviral vector has been reported by us as well as others [1,5,6,10,11,13,21]. Encouraged by these results, we first investigated the potential of a novel strategy for introducing exogenous Bcl-2 into ORNs by using the Cre/loxP recombina- tion system with an adenoviral vector [19]. The results presented here are expected to contribute to the development of clinical applications of the Bcl-2 gene transfer to the peripheral olfac- tory system for the treatment of olfactory dysfunction. The virus preparation of AxCALNLBcl-2 and AxCANCre has been described elsewhere [19]. Human Bcl-2 cDNA was kindly provided by Dr SJ Korsmeyer (Harvard Medical School, Boston, MA, USA). The viruses were grown in HEK 293 cells and purified by CsCl gradient centrifugation. Virus titer was 0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.01.068