We developed an expression profiling array to examine pro-apoptotic and pro-survival genes in dog retinal degeneration models. Gene-specific canine TaqMan assays were developed and included in a custom real-time quantitative reverse transcription-PCR (qRT-PCR) array. Of the 96 selected genes, 93 belonged to known relevant pro-apoptotic and pro-survival pathways, and/or were positive controls expressed in retina, while three were housekeeping genes. Ingenuity Pathway Analysis (IPA) showed that the selected genes belonged to expected biological functions (cell death, cell-mediated immune response, cellular development, function, and maintenance) and pathways (death receptor signaling, apoptosis, TNFR1 signaling, and induction of apoptosis by HIV1). Validation of the profiling array was performed with RNA extracted from cultured MDCK cells in the presence or absence of treatment with 10 μM staurosporin for 5 or 10 h. The vast majority of the genes showed positive amplifications, and a number of them also had fold change (FC) differences > ±3 between control and staurosporin-treated cells. To conclude, we established a profiling array that will be used to identify differentially expressed genes associated with photoreceptor death or survival in canine models of retinal degenerative diseases with mutations in genes that cause human inherited blindness with comparable phenotypes.
Dog model Retinal degenerative diseases RNA expression profiling array Real-time quantitative reverse transcription-PCR Pro-apoptosis genes Pro-survival genes Cell death Cell survival Madin–Darby canine kidney cells Staurosporin
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This study was supported by the Foundation Fighting Blindness (FFB), NIH Grants EY06855, 13132, and 17549, Fight for Sight Nowak Family Grant, the Van Sloun Fund for Canine Genetic Research, and Hope for Vision. The authors thank Rupa Gosh for technical assistance with qRT-PCR experiments.
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