Research Report High-purity selection and maintenance of gene expression in human neuroblastoma cells stably over-expressing GFP fusion protein Application for opioid receptors desensitization studies Benjamin Aguila 1 , Mikel Roussel, Philippe Jauzac, Stéphane Allouche ⁎ Laboratoire de Biologie cellulaire et moléculaire de la signalisation, UPRES-EA 3919, Université de Caen, France ARTICLE INFO ABSTRACT Article history: Accepted 18 July 2006 Available online 28 August 2006 Chronic use of opiates such as morphine is associated with drug tolerance, which is correlated with the desensitization of opioid receptors. This latter process involves phosphorylation of opioid receptors by G protein-coupled receptors kinases (GRKs) and subsequent uncoupling by β-arrestins. To explore these molecular mechanisms, neuronal cell lines, endogenously expressing the opioid receptors, provide an ideal cellular model. Unfortunately, there are two major drawbacks: (1) these cells are refractory to cDNA introduction, resulting in low transfection efficiency; (2) continuous culturing of transfected cells invariably leads to phenotypic drift of the cultures even after an antibiotic selection. So, these cells were dropped in favor of heterologous expression systems, which are easier to transfect but whose relevance as adequate cellular model for studying opioid receptor regulation should be questioned, as recently demonstrated by [Haberstock-Debic, H., Kim, K.A.,Yu, Y.J., von Zastrow, M., 2005. Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J. Neurosci. 25, 7847–7857]. In this work, we describe a method, based on fluorescence-activated cell sorting (FACS), to select and maintain a high proportion of transfected SK-N-BE cells (a neuronal cell line endogenously expressing human Delta–Opioid Receptor (hDOR)), expressing the β-arrestin1 fused to green fluorescent protein (GFP). While in functional experiments, we were not able to observe a major effect in non-sorted SK-N-BE cells expressing β-arrestin1–GFP, the enrichment by 18-fold with FACS resulted in a robust increase of β-arrestin1–GFP expression associated with strong hDOR desensitization. Moreover, this method also allows to counteract the phenotypic drift and to maintain a high-purity selection of SK-N-BE cells expressing β-arrestin1–GFP. Thus, this approach provides a valuable tool for exploring opioid receptors desensitization in neuronal cells. © 2006 Elsevier B.V. All rights reserved. Keywords: FACS GFP SK-N-BE hDOR β-arrestin1 Desensitization Abbreviations: hDOR, human delta opioid receptor FACS, fluorescence-activated cell sorting β-arr1–GFP, β-arrestin1–GFP BRAIN RESEARCH 1114 (2006) 11 – 18 ⁎ Corresponding author. Fax: +33231064985. E-mail address: allouche-s@chu-caen.fr (S. Allouche). 1 Recipient of a fellowship from the Conseil Régional de Basse-Normandie. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.07.069 available at www.sciencedirect.com www.elsevier.com/locate/brainres