Mouse models of overexpression of GCAP2 and loss-of-function of GCAP1 and GCAP2 used in the study.

A. GCAP2 transgene construct. MOP, 4.4 kb-version of the mouse opsin promoter; bGCAP2, cDNA of bovine GUCA1B gene encoding guanylate cyclase activator protein 2 (GCAP2); MP1pA, polyadenylation signal of mouse protamine gene 1. B. Western blot of total retinal homogenates illustrating GCAP2 level of expression in the GCAP2+ line. Equivalent fractions of a retina (1/10) of WT and GCAP2+ mice were resolved in a 12% SDS-PAGE, transferred to a nitrocellulose membrane and incubated with a polyclonal Ab anti-GCAP2. The bovine (transgenic) and murine (endogenous) isoforms of GCAP2 can be resolved on the basis of their 3-aa difference in size. In the GCAP2+ transgenic line bGCAP2 is expressed to 1.5-fold the endogenous GCAP2 expression [27]. C. Light micrographs of vertical sections of the retina of dark-reared WT, GCAP2+ and GCAP2+/+ (transgenic line bred to homozygosity, that expresses transgenic GCAP2 to 3-fold the endogenous GCAP2 level) at postnatal day 40. Mice overexpressing GCAP2 show at this age a normal retinal morphology. D. Expression of bGCAP2 transgene in the GCAP1/GCAP2 double knockout background (GCAPs−/− background). Western blot shows expression of transgenic bGCAP2 in the absence of endogenous GCAP2 in the GCAPs−/−GCAP2+ mice. E. Light micrographs of vertical sections of the retina from GCAPs−/− and GCAPs−/−GCAP2+ at 1, 3 or 5 months of age, when reared in standard cyclic light. Mice lacking GCAP1 and GCAP2 retain the normal thickness of outer nuclear layer, that is, the normal number of photoreceptor cells for up to 8 months of age. Mice in which GCAP2 expression is restored in the GCAPs−/− background do not show obvious signs of retinal degeneration at the light microscopy level.