The Insulitis Reporter Mouse

描述（由申请人提供）：1型糖尿病（T1DM）是一种T细胞介导的自身免疫性疾病，是最常见的儿童自身免疫性疾病。其主要病理是免疫细胞介导的对产生胰岛素的胰腺细胞的完全破坏。这个过程发生在白细胞浸润胰岛时，这个过程被称为胰岛素炎，它选择性地消耗产生胰岛素的β细胞。当这种缺陷变得严重时，坦率的糖尿病被观察到急性高血糖发作。T1DM的免疫病理在非肥胖糖尿病（NOD）小鼠中得到了很好的模拟，这已经成为自身免疫性糖尿病研究的“金标准”。在人类临床环境和NOD研究模型中，最令人烦恼的是缺乏亚临床胰岛素及其严重程度的明确指标，因为胰岛素过程是隐匿的，并且在完全没有明显症状的情况下持续很长一段时间。无法确定胰岛素的时间和分期，极大地阻碍了对T1DM病因和分子发病机制的研究。因此，迫切需要一种重要的、实时的胰岛素检测方法。为了解决这个问题，我们的目标是制造一种NOD小鼠，它通过分子报告来表明胰岛素的开始和严重程度，这种分子报告可以通过微创和非致命的方法容易、可靠和快速地检测到。该报告必须允许快速和具有成本效益的定性评估胰岛素。可溶性人胎盘碱性磷酸酶（sHPAP）是理想的候选酶。它在血液中具有良好的生物利用度和稳定性，具有独特的底物，现有的灵敏荧光法用于定量检测，并且对小鼠碱性磷酸酶抑制剂具有抗性。我们之前的工作，使用功能基因组学，已经确定Reg3gamma基因在NOD小鼠的胰岛素启动时经历快速和持续的上调。这种β细胞特异性基因在β细胞存活中具有保护和抗凋亡作用，在未浸润的胰岛中没有或非常低的基础表达，但在胰岛素炎中表现出高达30倍的渐进式表达。在这里，我们提出制造敲入NOD小鼠，使用内源性Reg3gamma启动子来驱动胰腺β细胞中sHPAP的表达。由于β细胞是与血液密切接触的特殊分泌细胞，我们假设在胰岛的自然免疫浸润下，β细胞会产生sHPAP。然后，我们可以从NOD小鼠的少量血液样本中量化sHPAP，以确定胰岛素炎的开始和严重程度。为此，我们提出以下具体目标：具体目标1：通过靶向引入可溶性人胎盘来生产胰岛素报告（InsuRe） NOD小鼠！碱性磷酸酶进入Reg3gamma位点。目的2：通过定性分析NOD小鼠的sHPAP表达和记录的胰岛素炎症，验证NOD小鼠的特异性和敏感性。

DESCRIPTION (provided by applicant): Type 1 diabetes mellitus (T1DM) is a T cell-mediated autoimmune disease, and the most prevalent pediatric autoimmune disorder. Its primary pathology is the complete immune cell-mediated destruction of the insulin-producing pancreatic beta cells. This process occurs upon the infiltration of the pancreatic islets by leukocytes, in a process termed insulitis, which selective depletes insulin-producing beta cells. When this deficient becomes critical, frank diabetes is observed as the acute onset of hyperglycemia. The immunopathology of T1DM is well modeled in the non- obese diabetic (NOD) mouse, which has emerged as the "gold-standard" of autoimmune diabetes research. What is most vexing in both the human clinical setting and the NOD research model, is the lack of a clear indicator of sub-clinical insulitis and its severity, as the insulitis process is occult and occurs over a prolonged period of time in the total absence of overt symptoms. The inability to time and stage insulitis has greatly hampered research into T1DM etiology and molecular pathogenesis. Therefore there is a pressing need for a vital and real-time measure for insulitis. To address this, our goal is to product an NOD mouse that signals the initiation and severity of insulitis by means of a molecular report that is readily, reliably and rapidly detectable by a minimally-invasive and non-lethal means. This reporter must allow for a rapid and cost-effective qualitative assessment of insulitis. Soluble human placental alkaline phosphatase (sHPAP) is an ideal candidate. It has excellent bio-availability and stability in the blood, it has a unique substrate, an existing and sensitive fluorimetric assay for quantitative detection, and it is resistant to inhibitors of mouse alkaline phosphatase. Our previous work, using functional genomics, has identified Reg3gamma as a gene that undergoes rapid and sustained up-regulation upon the initiation of insulitis in the NOD mouse. This beta cell-specific gene has a protective, anti-apoptotic role in beta cell survival and has no to very low basal expression in uninfiltrated islets but exhibits up to a 30-fold progressive increase in expression with insulitis. Here we proposed to make knock-in NOD mice that use the endogenous Reg3gamma promoter to drive the expression of sHPAP in pancreatic beta cells. As beta cells are specialized secretory cells with intimate access to the blood, we hypothesize that upon the natural immune-infiltration of islets, beta cells will produce sHPAP. We can then quantify sHPAP from a small blood sample of NOD mice to determine the initiation and severity of insulitis. To this end, we propose the following specific aims: Specific Aim 1: To product insulitis reporter (InsuRe) NOD mice by the targeted introduction of soluble human placenta! alkaline phosphatase into the Reg3gamma locus. Specific Aim 2: To validate the specificity and sensitivity of the insulitis reporter NOD mice by qualitative analysis of sHPAP expression and documented insulitis in NOD mice.