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.

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons

“…Previous reports have usually used transgenic constructs to label and observe more superficially located cells in the skin, heart, eye, muscle, or tail, where the opacity of X. laevis embryos presents less of a problem. (e.g., Moritz et al, 1999;Jansen et al, 2002;Lim et al, 2004;Smith et al, 2005;Scheenen et al, 2009;Yokoyama et al, 2011;Vivien et al, 2012;Haeri et al, 2013;Loots et al, 2013;Tam et al, 2013;Zhuo et al, 2013) [although also see (Love et al, 2011) and(L'Hostis-Guidet et al, 2009) as these authors used a few widely expressed neuronal promoters to test different types of transgenesis].…”

Retinal tissue preparation for high-resolution live imaging of photoreceptors expressing multiple transgenes

“…Live imaging of retinal tissues is a suitable method to study the transport, localization, interaction and dynamics of different proteins in highly specialized photoreceptors. This approach can be utilized to learn more about the pathogenesis of eye diseases and potential therapies [ 8 , 17 , [20] , [21] , [22] ]. These types of studies are not suited for fixed tissue imaging.…”

Modeling congenital kidney diseases in Xenopus laevis

“…Inducible systems have also been successfully used in Xenopus embryos, which allows for temporal or spatial control of gene expression. Four inducible systems were developed in Xenopus : the GAL4-UAS (Box 1) (Denayer et al, 2006), Tet-On (Box 1) (Rankin et al, 2011), heat-shock inducible (Roose et al, 2009) and the Dex-inducible (Box 1) strategy (Zhuo et al, 2013). In addition, knocking down a specific protein can be titrated, either by using morpholinos at varying doses to reduce lethality (Eisen and Smith, 2008) or by microinjecting less mRNA of an inhibitor of the protein of interest (Baker et al, 1999).…”