Abstract
Abstract: :
Purpose: Guanylyl cyclase activating protein, GCAP-1, that is highly expressed in cones and at lower concentrations in rods, activates retinal photoreceptor membrane guanylyl cyclase, retGC, at less than 100 nM Ca2+ (in the light) and inhibits retGC at higher than 500 nM Ca2+ (in the dark). Two mutations, Y99C and E155G, associated with dominant congenital cone degeneration in humans, make GCAP-1 active even when Ca2+ levels exceed 1 µM [1-3]. We attempted to express the mutant GCAP-1 in mouse rods at sufficiently high concentrations in order to develop animal model that would allow studying main physiological and biochemical changes caused by these mutations. Methods: Bovine GCAP-1 cDNA fragments were inserted in mouse genome under control of mouse rhodopsin promoter using injection into male pronuclei of B6/D2 hybrid mouse eggs. Mice that received the transgene as a result of random insertion were selected using PCR and DNA hybridization for further breeding on C57B6 genetic background and, as additional precaution, verified for the absence of the rd mutation. Transgene expression in mouse retinas was documented using immunoblot and immunofluorescence microscopy. Morphological analysis was performed using fixation by osmium at different ages. Results: We have developed three different lines of mice that carry the Y99C GCAP-1, in which the levels of the transgene protein expression were equal or up to 5-fold higher than the endogenous GCAP-1. We have also developed a control transgenic line that expressed normal bovine GCAP-1 at approx. 3- fold higher level than the endogenous mouse GCAP-1. Based on the initial morphological analysis, overexpresson of the Y99C mutant in transgenic mice results in retinal degeneration detectable at as early as 5 weeks of age. Photoreceptor death rapidly progresses in these mice so that after 3 months all photoreceptors, both rods and cones, completely degenerate. In contrast to that, no signs of retinal degeneration in mice that overexpress to a comparable level normal GCAP-1 were observed even at the age of 4.5 months. We have also produced several lines of mice that carry a transgene for the E155G GCAP-1. Conclusions: (1) Unlike wild type GCAP-1, overexpression of the constitutively active GCAP-1 mutant in rods can cause retinal degeneration. (2) These transgenic mice may provide a model for further study of the biochemical changes that associate with the constitutively active GCAP-1 in photoreceptors. References: [1] Dizhoor et al., J Biol Chem 273:17311-4., 1998, [2] Sokal et al., Mol Cell 2:129-33, 1998, [3] Wilkie et al. Am J Hum Genet 69:471-80, 2001
Keywords: 561 retinal degenerations: cell biology • 517 photoreceptors • 606 transgenics/knock-outs