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N Mori, S Kubota, A Kobayashi, T Higashide, MJ McLaren, G Inana; Construction of a Knock-out Model of HRG4(UNC119) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1379.
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Purpose: HRG4 is a novel photoreceptor protein we have isolated by a differential cloning approach (Higashide et al., J. Biol. Chem. 271:1797-1804,1996). Immunofluorescence microscopy and immunocytochemistry localized HRG4 in the rod and cone photoreceptor synapses, establishing it as the first synaptic protein enriched in the photoreceptors (Higashide et al., Invest. Ophthalmol. Vis. Sci. 39:690-698,1998). The HRG4 gene has been mapped to chromosome 17q11.2 and shown to consist of five exons (Higashide et al, Genomics. 57:446-450, 1999). A premature termination codon mutation in HRG4 was uncovered in a patient with late-onset cone-rod dystrophy, and transgenic mice expressing the identical mutation demonstrated a late-onset reduction in b-wave by ERG and retinal degeneration with marked synaptic degeneration (A. Kobayashi et al., Invest. Ophthalmol. Vis. Sci. 41:690-698,2000). The analysis of changes in retinal synaptic proteins in the HRG4 transgenic mice demonstrated a decrease in synaptic vesicle associated proteins and an increase in cytoplasmic and plasma membrane proteins in the older transgenic retina (Kubota et al., Invest. Ophthalmol. Vis. Sci. in press). We are constructing a knock-out model of HRG4 in order to further investigate the function and pathogenicity of HRG4. Methods: A knock-out targeting vector which interrupts exon 2 of the HRG4 gene with a stop codon mutation and a neomycin resistance gene was constructed and transfected into mouse ES cells. The successfully targeted ES cells were used for blastocyst injection to make chimeras, and the chimeras were mated with normal mice to produce heterozygotes. Results: Three lines of successfully targeted ES cells were obtained (189, 95, 43) and were used for blastocyst injection. A total of 11 chimeras were produced, and breeding of the #95 chimeras produced heterozygously targeted mice. Conclusion: Germline transmission of the targeted HRG4 gene was achieved in the form of heterozygous offspring. The HRG4 knock-out model, both heterozygous and homozygous, should be an important resource for understanding the function and pathogenicity of HRG4.
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