Functional Analysis of Rds/peripherin in Disc Morphogenesis Using Tet–Off Rds–Transgenic Mice on an rds–/– Mutant Background

Abstract

Abstract: : Purpose:Rds/Peripherin–2 gene is a member of a Tetraspanin family of Transmembrane Proteins and is responsible for many of currently identified and studied Retinopathies in human eye diseases. Deletion or mutation in this gene greatly affects disc morphogenesis. In order to better understand the role that Rds plays in disc formation, we set up to create binary transgenic mice with Tetracycline–inducible Switch that turns off the Expression of Rds gene expression at will. This effect, we hope, can allow us to gain insight into the initial Stage where Rds role is critical for disc morphogenesis. Methods: Tet–off double Transgenic DNA was constructed to stably express a fusion protein of the Tet repressor and the herpes simplex virus VP16 (tTA3) downstream of a mouse rhodopsin–promoter. This in turn, acts like a switch by binding to the Tetracycline–controlled transactivator (TRE) which activates the transcription of the Rds gene. The binding can be blocked by the administration of Doxycycline. Results: Out of 120 pups born, 20 were positive for the double transgene and only 16 were positive for one or the other transgene. Mice carrying the binary transgenes were mated with a null mutant Rds(–/–) mice and offsprings collected and analyzed for the presense of the double transgenes by Polymerase Chain Reaction. First Strand cDNA sysnthesis following RT–PCR reveals the presense of the mRNA transcript specific for each of the tTA3 and the mRDS constructs. The Expression of these constructs could only be detected in retina tissues but not in other tissues tested by RT–PCR method. Conclusions: After administering Doxycycline to these animals, rds expression should be sharply reduced. The morphology could be tested under a high resolution Electron Microscopy. Considering the rate at which rds shading takes place at 10% every 24 hrs, and upon examination of the ultra structural cross–sections of the retina, we may learn more about the role of rds and its implication it has on the morphology, disc alignment, rate of addition, and shedding of outer–segment discs.

Keywords: photoreceptors • protein structure/function • retinal degenerations: cell biology