title: High-purity selection and maintenance of gene expression in human neuroblastoma cells stably over-expressing GFP fusion protein. Application for opioid receptors desensitization studies.
abstract: 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 beta-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 beta-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 beta-arrestin1-GFP, the enrichment by 18-fold with FACS resulted in a robust increase of beta-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 beta-arrestin1-GFP. Thus, this approach provides a valuable tool for exploring opioid receptors desensitization in neuronal cells.
authors.affiliation: Laboratoire de Biologie cellulaire et moléculaire de la signalisation, UPRES-EA 3919, Université de Caen, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Aguila
authors.forename: Mikel
authors.initials: M
authors.lastname: Roussel
authors.forename: Philippe
authors.initials: P
authors.lastname: Jauzac
authors.forename: Stéphane
authors.initials: S
authors.lastname: Allouche
mesh_terms: D000704:Analysis of Variance; D019390:Arrestins / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D004361:Drug Tolerance / Q000502:physiology*; D005048:Etorphine / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods; D009294:Narcotics / Q000494:pharmacology; D009447:Neuroblastoma; D011957:Receptors, Opioid / Q000187:drug effects / Q000502:physiology*; D012641:Selection, Genetic; D013997:Time Factors; D014162:Transfection / Q000379:methods; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D009294:Narcotics; D011957:Receptors, Opioid; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D005048:Etorphine; D000242:Cyclic AMP