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R. Ufret-Vincenty, A. McMahon, P. Chen, J. Y. Niederkorn, W. Kedzierski; Generation and Characterization of Chimeric Complement Factor H Transgenic Mice With an "At-Risk" Mutation for Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2008;49(13):210.
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Complement factor H (Cfh) is a key regulator of the alternative complement pathway. A single nucleotide polymorphism that leads to a histidine instead of a tyrosine at amino acid position 402 of Cfh (Y402H) increases the risk for age-related macular degeneration (AMD) up to 7-fold. Our goal is to generate transgenic mice expressing either the tyrosine or the histidine variants of Cfh in order to: (a) identify the molecular basis for the susceptibility to AMD observed in human H-CFH carriers, and (b) generate a relevant model of AMD that would allow the dissection of the role of the different arms of the immune system in the disease.
CFH consists of 20 short consensus repeats (SCR). Y402H localizes to SCR7. We have generated two transgenic constructs which consist of the human SCR6-8 sequence flanked by the mouse SCR1-5 and SCR9-20 sequences. One of the constructs codes for a tyrosine (Y-Cfh) at amino acid position 402, and the second one codes for a histidine (H-Cfh) at this position. The ApoE promoter was used to induce liver expression of the chimeric Cfh molecules and secretion into the circulation. The coding sequences were isolated and injected into C57Bl/6 fertilized eggs.
Sequencing validated the accuracy of our constructs. Genotyping allowed us to identify 7 transgenic founders for the H-Cfh transgene and 10 for the Y-Cfh transgene. A nuclease protection assay was used to determine levels of chimeric vs. native mRNA. We then used a western blot to determine the level of Cfh protein expression in the serum.
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