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S.A. Dilks, E.M. Stone, E.A. Pierce; Haploinsufficiency of Efemp1/Fibulin 3 Does Not Cause Drusen . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5282.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The dominantly inherited macular degenerations Doyne honeycomb retinal dystrophy (DHRD)/Malattia Leventinese (ML) are caused by a single mutation, Arg–345 to Trp (R345W), in the EFEMP1 (Fibulin 3) gene. These conditions are characterized by drusen formation and progressive visual loss, with similarities to age–related macular degeneration (AMD). The clinical similarity between DHRD/ML and AMD has lead to the suggestion that DHRD/ML could be a good model system for studying AMD. To create a mouse model of DHRD/ML we used gene targeting techniques to create Efemp1–R345W knockin mice. To investigate the effect of decreased Efemp1 levels, we have characterized the Efemp1–Neo–R345W mice produced in the process of generating the knockin mice. Methods: Mouse embyronic stem (ES) cells were transfected with a gene targeting vector designed to introduce the R345W mutation into exon 9 of the mouse Efemp1 gene. This vector contained a floxed neomycin selection cassette between exons 8 and 9. Six clones with the correctly targeted allele (Efemp1–Neo–R345W) were identified. Two of these clones were injected into C57BL/6 blastocysts to create chimeric mice. Chimeras were backcrossed with C57BL/6 mice to generate heterozygous Efemp1–Neo–R345W mice. F1 mice were intercrossed to generate homozygous mice. Mice were examined for the presence of drusen by indirect ophthalmoscopy and histology. The levels of Efemp1 mRNA were measured by quantitative real–time RT–PCR using RNA from retina and other tissues. Results: Twenty highly chimeric mice were generated from the Efemp1–Neo–R345W ES cells, and germline transmission of the mutant allele was observed. Heterozygous and homozygous mice are healthy and fertile. No evidence of drusen was detected in heterozygous mice up to 5 months of age or homozygous mice up to 3 months of age. Quantitative RT–PCR demonstrated that Efemp1 mRNA levels were decreased to approximately 54% of normal in heterozygous mice, and 10% of normal in homozygous mice. Discussion: The decreased Efemp1 mRNA levels observed in the Efemp1–Neo–R345W mice suggests that the selection cassette interferes with expression of the mutant mRNA, creating a hypomorphic allele. Lack of phenotype in these mice implies that the drusen observed in patients with DHRD/ML are caused by dominant effect of the R345W mutant EFEMP1, although additional evaluation of older animals is warranted. We will test this hypothesis by evaluating the phenotype of the Efemp1–R345W knockin mice following removal of the selection cassette.
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