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Michelle Reed, Li Jiang, Wolfgang Baehr; Creation and Characterization of a Bardet-Biedl Syndrome (BBS) Mouse Model Containing Mutations in Sdccag8 Using CRISPR/Cas9. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2257. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in Serologically defined colon cancer antigen 8 (SDCCAG8, NPHP10) are shown to be causative of Bardet-Biedl Syndrome (BBS), but the mechanism of retinal degeneration in these patients remains unknown. We are studying the mechanism of the retinal degeneration present in a Sdccag8 knockout mouse.
We performed confocal immunohistochemistry with anti-SDCCAG8 specificity. Our mutant mouse was created using CRISPR/Cas9 technology. We designed CRISPR guide RNAs (sgRNA) to target Exon 7 of Sdccag8. Patient DNA provided the mutations R231X (691-692 AG>TA) and AY236X (708 C>G) as a goal, resulting in a nonsense mutation. To generate the mutant mouse, 200 wild-type (WT) C57Bl/6 mouse embryos were injected with the sgRNA, template DNA and Cas9 protein. Genomic DNA was extracted from 17 pups and mutations were screened for by high-resolution melting analysis (HRMA) of Exon 7 PCR amplification.
SDCCAG8 is located in the basal body/connecting cilium area in the distal inner segment of WT photoreceptors. HRMA showed mutations in 6 mice and subsequent genotyping revealed small insertion/deletions (indels) in Exon 7. One indel (mut, 702_707delCACAT) was chosen due to the resulting truncation being within our window of inclusion (±5 amino acids compared to template mutation). Breeding with WT mice resulted in germline transmission and heterozygotes are being bred to generate the SDCCAG8mut/mut mouse line.We will be using OCT, Optomotry, ERG and confocal microscopy to assess photoreceptor function and to document progression of degeneration at P14, P30 and P50 using 5 mice each of SDCCAG8mut/+ and SDCCAG8mut/mut. SDCCAG8mut/mut. Immunocytochemical (ICC) staining of proteins involved in the phototransduction cascade (opsins, transducin, PDE) will be conducted to show possible protein mistrafficking.
We were successful in the generation of our mutant mouse and we anticipate results that phenocopy syndromic BBS. Through this model, we will learn about the function of SDCCAG8 in photoreceptors and the mechanism of SDCCAG8-dependent photoreceptor degeneration. The long-term goal is to design AAV gene replacement vectors for somatic gene therapy.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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