Genetic engineering of the Idd3 type 1 diabetes locus

描述（由申请人提供）： 在非肥胖糖尿病（NOD）小鼠（一种人类疾病的自发动物模型）中，已经鉴定了30多个自身免疫性1型糖尿病（T1 D）易感基因座（称为Idd）。其中，Idd 3基因座已被定位到3号染色体上的650 kb区域，包含5个蛋白质编码基因和2个预测基因，其中IL-2和IL-21是最佳候选基因。C57 BL/6（B6）衍生的Idd 3赋予强的T1 D抗性，而NOD衍生的间隔有助于疾病发展。值得注意的是，IL-2/IL-21基因座也已在全基因组关联研究中被定位用于人类T1 D。由于IL-2和IL-21基因之间的紧密连锁（相距100 kb），分别仅携带B6衍生的IL-2或IL-21基因的同源菌株还不能用于独立地测试它们中的一个或两个是否代表Idd 3潜在基因。最初的研究，涉及单倍型定位和表达分析提供的证据支持IL-2的Idd 3基因。最近，也出现了白细胞介素（IL）-21的致糖尿病作用，并且与NOD.Idd3B6同源株相比，NOD小鼠表达更高水平的这种细胞因子。由于IL-2和IL-21基因的等位基因变异存在于NOD和B6菌株之间，因此不能排除这两个基因都对Idd 3基因座的T1 D调节功能有贡献的可能性。然而，以前的研究缺乏适当的遗传工具来独立和明确地测试IL-2和/或IL-21是否是Idd 3区域内的偶然基因。在本申请中，我们提出使用锌指核酸酶（ZFN）建立小鼠模型系统，其中一对F1杂交株分别仅表达候选基因的两个亲本等位基因中的一个，但在整个基因组中是遗传上相同的，以直接确定所涉及的疾病基因的作用。ZFN技术特异性敲除缺乏生殖系可传递胚胎干细胞的小鼠品系中的基因的能力将使我们能够靶向标准NOD小鼠和NOD.Idd3B6同源品系中的IL-2和IL-21。我们的目标是通过建立一系列（NOD x NOD.Idd3B6）F1菌株来确定IL-2或IL-21或两者是否是Idd 3潜在基因，其中仅表达IL-2或IL-21的一个亲本等位基因（NOD或B6）。

DESCRIPTION (provided by applicant): Project Summary More than 30 autoimmune type 1 diabetes (T1D) susceptibility loci (termed Idd) have been identified in the nonobese diabetic (NOD) mouse, a spontaneous animal model for the human disease. Among those, the Idd3 locus has been mapped to a 650 kb region on Chromosome 3 containing five protein-coding and two predicted genes, of which Il-2 and Il-21 are top candidates. The C57BL/6 (B6)-derived Idd3 confers strong T1D resistance, whereas the NOD-derived interval contributes to disease development. Remarkably, the IL-2/IL-21 locus has also been mapped for human T1D in genome-wide association studies. Due to the tight linkage between Il-2 and Il-21 genes (100kb apart), congenic strains that respectively carry only B6-derived Il-2 or Il-21 gene have not been made available to independently test if either or both of them represent the Idd3 underlying genes. Initial studies that involved haplotype mapping and expression analyses provided evidence to support Il-2 as the Idd3 gene. Recently, a diabetogenic role of interleukin (IL)-21 has also emerged, and a higher level of this cytokine was shown to be expressed by NOD mice compared to the NOD.Idd3B6 congenic strain. Since allelic variations in both Il-2 and Il-21 genes exist between NOD and B6 strains, a possibility tha both genes contribute to the T1D regulatory function of the Idd3 locus cannot be excluded. However, previous studies lacked the appropriate genetic tools to independently and definitively test if Il-2 and/or Il-21 are the casual genes within the Idd3 region. In this application, we propose to use zinc-finger nucleases (ZFNs) to establish a mouse model system where a pair of F1 hybrid strains respectively express only one of the two parental alleles of a candidate gene, but are genetically identical throughout the genome to directly determine the role of the implicated disease gene. The ability of the ZFN technology to specifically knock out a gene in mouse strains lacking germ-line transmittable embryonic stem cells will allow us to target both Il-2 and Il-21 in standard NOD mice and the NOD.Idd3B6 congenic strain. Our goal is to determine if Il-2 or Il-21, or both are the Idd3 underlying genes by establishing a series of (NOD x NOD.Idd3B6) F1 strains where only one parental allele of Il-2 or Il-21 (NOD or B6) is expressed.