Figure 1A. Schematic of the targeting vector. In the vector, the RIBEYE-specific A-domain (yellow) is fused to the genetically encoded Ca2+-sensor GCamP3 (green), followed by a stop codon and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE, blue) and an PGK-neomycin resistance cassette (orange) that is flanked by frt-sites (red ovals). This entire DNA sequence is flanked by loxP-sites (dark red triangles). After homologous recombination into the RIBEYE locus (RBEKI+neo), Flp-recombinase (introduced via transgenic expression) removes the selection cassette to generate the RBEKI allele. Subsequently, Cre-recombinase excises the entire A-domain and its accompanying sequences to produce the KO allele (RBEKO). Selected restriction sites are shown. B. RIBEYE mutations do not alter mouse survival. Data show surviving post-weaning mice (at >P21) derived from matings between mice heterozygous for the RBEKI allele (dark red) or the RBEKO allele (bright red) normalized for the total number of mice (absolute numbers per genotype/absolute number of all mice per respective breeding strategy) The expected Mendelian ratio is indicated by the dashed line. Statistical analysis using the 2-test revealed no statistical difference between wild-type and mutant mice (RBEKI allele p=0.33; RBEKO allele p=0.11). C. Representative immunoblot of retina homogenates from homozygous RBEWT/WT, RBEKI/KI and RBEKO/KO mice probed with antibodies to CtBP2 (to detect the B-domain of RIBEYE and CtBP2) and to EGFP (to detect GCamP3 in the RIBEYE A-domain GCamP3 fusion protein referred to as RIB-G3). D. Measurements by RT-PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild-type mice (RBEWT/WT), KI-mice expressing the RIBEYE-A-domain fusion protein with GCamP3 (RBEKI/KI), and KO mice (RBEKO/KO). These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina, and to examine whether the RIBEYE A-domain KO impairs CtBP2 expression in brain, which expresses little RIBEYE. Assays are based on a qRT-PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms (RIBEYE and CtBP2). Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (***, p<0.001; n=4 mice per genotype). E. Representative immunofluorescence labeling of cryostat sections from littermate wild-type and RBEKI/KI mice for EGFP (green, to detect GCamP3 in RIB-G3) and SV2 (red, to label synapses) demonstrates expression of the A-domain/GCamP3 fusion protein in knockin mice (OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, granule cell layer). Scale bar = 5 µm. F. Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT/WT and RBEKO/KO mice. Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies; results are normalized to loading controls depending on molecular weights (actin, fodrin, VCP, and GDI) and to wild-type controls. Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (*, p<0.05; n=4 mice per genotype). G. Immunofluoresence labeling for CtBP2 (green, to label RIBEYE) and mGluR6 (red, to label photoreceptorsynapses) in cryostat sections from littermate RBEWT/WT and RBEKO/KOmouseretinas demonstrates that the KO abolishes RIBEYE expression (OPL, outer plexiform layer). Scale bars: 5 µm.