An inducible expression system to measure rhodopsin transport in transgenic Xenopus rod outer segments

PLoS One. 2013 Dec 6;8(12):e82629. doi: 10.1371/journal.pone.0082629. eCollection 2013.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Cell Line
  • Cells, Cultured
  • Dexamethasone / pharmacology
  • Gene Expression Regulation / drug effects
  • Gene Expression*
  • Genes, Reporter
  • Humans
  • Protein Transport
  • Reproducibility of Results
  • Rhodopsin / genetics*
  • Rhodopsin / metabolism*
  • Rod Cell Outer Segment / metabolism*
  • Transgenes
  • Xenopus

Substances

  • Dexamethasone
  • Rhodopsin