Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Keywords: age-related macular degeneration • inflammation • pathobiology