IDENTIFICATION OF GENES REGULATING ARTHRITIS IN RATS

胶原诱导的关节炎是一种成熟的关节炎实验动物模型，已用于深入了解类风湿性关节炎的发病机制。 这项建议的总体目标是确定非MHC基因，调节这种形式的实验性关节炎的发展。 在导致本提案的实验中，利用了这样一个事实，即两个近交系大鼠品系共享相同的MHC单倍型，RT1av1，在其对同源大鼠II型胶原诱导的关节炎的易感性方面存在显著差异，尽管两个品系都表现出胶原自身抗体的产生。关节炎易感DA和关节炎抗性ACI大鼠之间的F2杂交中的全基因组扫描确定了大鼠2号染色体上的一个非MHC DA起源的数量性状位点（QTL）Cia7，以及2号和10号染色体上的另外两个暗示性位点。 还发现了显著的性别效应。 这三个基因座映射到基因组间隔同线的区域先前确定的小鼠和人类基因组中含有的基因参与不同形式的自身免疫性疾病的易感性，进一步强调了本研究的相关性。 该建议假设上述三个QTL含有将胶原免疫诱导的无症状自身免疫状态转化为明显自身免疫疾病的基因。 第一个目标是构建基因型引导的速度同源大鼠，将DA片段引入ACI背景，反之亦然。 将在同类动物中研究与这三个QTL的性别互作。 在后期阶段，将产生多同源大鼠以确定三个基因座的相互作用。 第二个目标是识别基因。 为了实现这一目标，将使用两种基本方法。 首先，将采用候选基因策略，利用小鼠和人类基因组同源性作图数据，筛选候选基因外显子的突变，然后在适用时进行测序。 将在从滑膜组织和T细胞产生的cDNA文库中分析某些候选基因的差异表达。 第二种策略将遵循更标准的方法，首先通过产生重组体将同源间隔缩小到1 cM，然后分离该区域的转录本，克隆和测序。 这两种策略都将通过鉴定参与应答的候选替代免疫亚表型来补充，所述应答应提供对基因作用机制的洞察和免疫表型的替代标记，从而促进基因发现过程。

Collagen-induced arthritis is a well-established experimental animal model of arthritis that has been used to gain insight into the pathogenesis of rheumatoid arthritis. The overall objective of this proposal is to identify the non-MHC genes that regulate development of this form of experimental arthritis. In experiments that led up to present proposal, advantage was taken of the fact that two inbred rat strains sharing the same MHC haplotype, RT1av1, differed strikingly in their susceptibility to arthritis induced with homologous rat type II collagen, although both strains exhibited the production of collagen autoantibodies. A genome-wide scan in a F2 cross between arthritis-susceptible DA and arthritis-resistant ACI rats identified one non-MHC DA- originated quantitative trait locus (QTL), Cia7, on rat chromosome 2, and two other suggestive loci on chromosome 2 and 10. A significant sex effect was also identified. These three loci map to genomic intervals syntenic to regions previously identified in the mouse and human genomes containing genes involved in the susceptibility to different forms of autoimmune diseases, further emphasizing the relevance of this study. This proposal hypothesizes that the three QTL described above contain genes that act to convert the state of asymptomatic autoimmunity induced by the collagen immunization into a frank autoimmune disease. The first aim is construction of genotype-guided speed congenic rats, introducing DA segments into ACI background, and vice-versa. Sex interaction with the three QTL will be studied in the congenic animals. At later stages, polycongenic rats will be generated to determine the interaction of the three loci. The second aim is identification of the genes. In order to accomplish this goal, two basic approaches will be used. First, a candidate gene strategy will be employed, taking advantage of mouse and human genome homology mapping data, and screening candidate gene exons for mutations, followed by sequencing, when applicable. Differential expression of certain candidate genes will be analyzed in cDNA libraries generated from synovial tissue and T cells. The second strategy will follow a more standard approach, first narrowing the congenic interval to 1 cM through generation of recombinants, followed by isolation of transcripts in the regions, cloning and sequencing. Both of these strategies will be supplemented by identification of candidate surrogate immunologic sub-phenotypes involved in the response that should provide both insight into the mechanism of gene action and alternative markers for immunologic phenotypes thus facilitating the gene discovery process.