The Insulitis Reporter Mouse

胰岛炎报告鼠

• 批准号: 7134619
• 负责人: JONATHAN David KATZ
• 金额: $ 22.5万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7134619
• 关键词: NOD mouse; alkaline phosphatase; biomarker; disease /disorder classification; early diagnosis; embryonic stem cell; gene expression; gene targeting; genetic models; genetic recombination; human genetic material tag; insulin dependent diabetes mellitus; model design /development; pancreatic islet disorder; pancreatic islet function; pathologic process; reporter genes

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.

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，也是最常见的儿科自身免疫性疾病。其主要病理学是免疫细胞介导的对产生胰岛素的胰腺β细胞的完全破坏。这一过程发生在白细胞浸润胰岛时，这一过程称为胰岛素炎，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，明显的糖尿病被观察为高血糖的急性发作。 T1DM 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，该小鼠已成为自身免疫糖尿病研究的“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炎及其严重程度的明确指标，因为胰岛素炎过程是隐匿性的，并且在完全不存在的情况下会在很长一段时间内发生的明显症状。无法确定胰岛素炎的时间和分期极大地阻碍了对 T1DM 病因学和分子发病机制的研究。因此，迫切需要针对胰岛素炎采取重要且实时的措施。为了解决这个问题，我们的目标是生产一种 NOD 小鼠，通过分子报告来发出胰岛素炎的发生和严重程度的信号，该分子报告可以通过微创和非致命的方式轻松、可靠和快速地检测到。该报告者必须能够对胰岛素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，具有独特的底物、现有的灵敏的定量检测荧光测定方法，并且对小鼠碱性磷酸酶抑制剂具有抵抗力。我们之前的工作使用功能基因组学，已确定 Reg3gamma 是一种在 NOD 小鼠出现胰岛素炎时快速持续上调的基因。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基本表达为零或非常低，但在胰岛炎时表达逐渐增加 30 倍。在这里，我们建议制作敲入型 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 细胞是特殊的分泌细胞，可以密切接触血液，我们假设在胰岛自然免疫浸润后，β 细胞将产生 sHPAP。然后我们可以从 NOD 小鼠的少量血液样本中定量 sHPAP，以确定胰岛素炎的发生和严重程度。为此，我们提出以下具体目标： 具体目标1：通过定向引入可溶性人胎盘，生产胰岛素报告基因（InsuRe）NOD小鼠！碱性磷酸酶进入 Reg3gamma 基因座。具体目标 2：通过对 NOD 小鼠中 sHPAP 表达和记录的胰岛素炎进行定性分析，验证胰岛素报告基因 NOD 小鼠的特异性和敏感性。