The Insulitis Reporter Mouse

描述（由申请人提供）：1型糖尿病（T1 DM）是一种T细胞介导的自身免疫性疾病，是最常见的儿科自身免疫性疾病。其主要病理是产生胰岛素的胰腺β细胞的完全免疫细胞介导的破坏。这一过程发生在白细胞浸润胰岛时，称为胰岛炎，选择性消耗产生胰岛素的β细胞。当这种缺陷变得严重时，观察到明显的糖尿病是高血糖症的急性发作。T1 DM的免疫病理学在非肥胖糖尿病（NOD）小鼠中得到了很好的建模，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炎及其严重程度的明确指标，因为胰岛炎过程是隐匿的，并且在完全没有明显症状的情况下长时间发生。由于无法对胰岛炎进行时间和分期，极大地阻碍了对T1 DM病因学和分子发病机制的研究。因此，迫切需要一个重要的和实时的措施，胰岛炎。为了解决这个问题，我们的目标是生产一种NOD小鼠，其通过分子报告的方式发出胰岛炎的开始和严重程度的信号，该分子报告可以通过微创和非致命的手段容易、可靠和快速地检测。该报告者必须允许对胰岛炎进行快速且具有成本效益的定性评估。可溶性人胎盘碱性磷酸酶（sHPAP）是理想的候选者。它在血液中具有优异的生物利用度和稳定性，它具有独特的底物，现有的和灵敏的荧光定量检测方法，并且它对小鼠碱性磷酸酶抑制剂具有抗性。我们以前的工作，使用功能基因组学，已确定Reg 3gamma作为一个基因，经历了快速和持续的上调后，开始在NOD小鼠胰岛炎。这种β细胞特异性基因在β细胞存活中具有保护性、抗凋亡作用，并且在未浸润的胰岛中没有至非常低的基础表达，但在胰岛炎中表现出高达30倍的表达进行性增加。在这里，我们提出了使用内源性Reg 3 γ启动子来驱动胰腺β细胞中sHPAP的表达的敲入NOD小鼠。由于β细胞是与血液密切接触的特化分泌细胞，我们假设在胰岛的天然免疫浸润后，β细胞将产生sHPAP。然后，我们可以从NOD小鼠的少量血液样品中定量sHPAP，以确定胰岛炎的起始和严重程度。为此，我们提出了以下具体目标：具体目标1：通过可溶性人胎盘的靶向导入来生产胰岛炎报告基因（InsuRe）NOD小鼠！将碱性磷酸酶导入Reg 3 γ基因座。具体目标二：通过对NOD小鼠中sHPAP表达和记录的胰岛炎进行定性分析，验证胰岛炎报告基因NOD小鼠的特异性和灵敏度。

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.