Abstract
Abstract: :
Purpose: Retinitis Pigmentosa (RP) 18 is one of 3 forms of RP caused by mutations in pre–RNA splicing factors, which as a group constitute the second leading cause of RP. A missense mutation (T494M) in PRPF3 causes RP18 through an unknown mechanism. It is not known how mutations in the ubiquitous PRPF3 can cause retina specific disease. We have generated Prpf3 knockout mice to study the disease mechanism of RP18. Methods: We searched the BayGenomics Gene Trap Consortium (http://baygenomics.ucsf.edu/) for an ES cell line with a disruption of Prpf3. We obtained clone RRO–284, which has the pGT2lxf gene trap vector inserted into the Prpf3 gene just after exon 2 based on 5’ RACE data from BayGenomics. We injected the RRO–284 ES cells into blastocysts to generate chimeric founder mice. The amount of Prpf3 protein in the retina was assessed by Western blotting using anti–Prpf3 antibodies. The Prpf3 signals on the blots were normalized to those of tubulin. Visual function of F1 mice heterozygous for the gene trap allele was evaluated by electroretinograph (ERG) analysis. Results: We confirmed that the genetrap insertion disrupts the Prpf3 locus by Southern blot analysis. Germline transmission of the genetrap allele was achieved from 5 of 8 chimeric founders tested. To date, all heterozygous Prpf3–Δ284 mice are healthy and fertile. Preliminary data from ERG analyses suggests a 25% decrease in visual function at 8 weeks of age in Prpf3–Δ284 heterozygotes compared to littermate controls. No changes in retinal histology have been identified to date. Western blot analysis demonstrates a decrease of approximately 50% in Prpf3 protein in heterozygous mice compared to littermate controls. Conclusions: The Δ284 gene trap insertion appears to create a null Prpf3 allele based on the decrease in Prpf3 protein observed in the retinas of Prpf3–Δ284 mice. The finding that Prpf3–Δ284 heterozygous mice are healthy and fertile confirms retina–specific nature of the pathogenicity of mutations in PRPF3. The ERG data indicate that haploinsufficiency of Prpf3 can result in retinal degeneration. This suggests that the T494M mutation in human PRPF3 may disrupt protein function. Continued evaluation of this line and comparison to Prpf3–T494M–knockin mice which we have also generated is needed to determine how mutations in PRPF3 cause retinal disease.
Keywords: retinitis • transgenics/knock-outs