title: An inducible expression system to measure rhodopsin transport in transgenic Xenopus rod outer segments.
abstract: We developed an inducible transgene expression system in Xenopus rod photoreceptors. Using a transgene containing mCherry fused to the carboxyl terminus of rhodopsin (Rho-mCherry), we characterized the displacement of rhodopsin (Rho) from the base to the tip of rod outer segment (OS) membranes. Quantitative confocal imaging of live rods showed very tight regulation of Rho-mCherry expression, with undetectable expression in the absence of dexamethasone (Dex) and an average of 16.5 µM of Rho-mCherry peak concentration after induction for several days (equivalent to >150-fold increase). Using repetitive inductions, we found the axial rate of disk displacement to be 1.0 µm/day for tadpoles at 20 °C in a 12 h dark /12 h light lighting cycle. The average distance to peak following Dex addition was 3.2 µm, which is equivalent to ~3 days. Rods treated for longer times showed more variable expression patterns, with most showing a reduction in Rho-mCherry concentration after 3 days. Using a simple model, we find that stochastic variation in transgene expression can account for the shape of the induction response.
authors.affiliation: Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States of America.
authors.forename: Xinming
authors.initials: X
authors.lastname: Zhuo
authors.affiliation: Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States of America.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Haeri
authors.affiliation: Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States of America.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Solessio
authors.affiliation: Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States of America.
authors.forename: Barry E
authors.initials: BE
authors.lastname: Knox
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D021381:Protein Transport; D015203:Reproducibility of Results; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D019076:Transgenes; D014981:Xenopus
chemical_list: D003907:Dexamethasone; D012243:Rhodopsin