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.
Highlights
Xenopus photoreceptors have played an important role in understanding the cell biology of membrane assembly [1,2,3,4], retinal disease [5,6,7] and ciliary transport [8]
In the absence of Dex, cells transfected with either pCMV:eGFP or pCMV:G3 had no significant luciferase activity compared to untransfected cells
Since we intend to utilize this system in transgenic Xenopus, which are housed at lower temperatures, we measured the rate at 27 °C (Figure 1C and 1D) and found that it is 2.2-fold slower than at 37 °C
Summary
Xenopus photoreceptors have played an important role in understanding the cell biology of membrane assembly [1,2,3,4], retinal disease [5,6,7] and ciliary transport [8]. The photoreceptors develop rapidly, forming light-sensitive outer segment (OS) membranes within a week post-fertilization [12,13] and expression of fluorescently tagged proteins are readily expressed [14]. The OS contains high concentrations (~3 mM) of the integral membrane protein rhodopsin [15], which is synthesized in the inner segment and rapidly delivered to the base of the OS for incorporation into disk membranes [16,17,18,19,20]. The distance along the OS axis from the base is linearly related to the time elapsed since incorporation for 4-6 weeks and offers an opportune model to investigate membrane protein synthesis and processing
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