Studies of Collagen Gene Regulation in Two Murine Models

描述（由申请人提供）：本提案将使用两个鼠标 突变是硬皮病、紧皮肤（Tsk）和紧皮肤2的模型 （Tsk2）.这两种突变都显示胶原蛋白和其他 皮肤中的细胞外基质成分，这是人类皮肤的标志性特征， 疾病拟议研究的长期目标是利用 这两种突变与几系转基因小鼠相结合， 用于剖析疾病发病机制的分子机制的实验工具。 提出了具体的实验，用于鉴定参与的基因， 真皮成纤维细胞细胞外基质合成的调节。两 已规划实验战略，并将其纳入三个具体目标。 具体目标1侧重于鉴定I型糖尿病中的顺式作用元件 突变体中增加Collal mRNA产生所需的胶原基因 真皮成纤维细胞定义“纤维化”的具体要素将提供一个基础 用于鉴定与这些DNA相互作用的交易因子， 增加胶原蛋白基因表达。这些元素将被定义为 通过研究携带各种基因的Collal CAT报告基因的表达， 5'启动子区的片段以及第一启动子区的特异性缺失， 内含子。将在皮肤样品中评估每种转基因的表达 分离自Tsk、Tsk 2和正常小鼠。此外，转基因表达将是 在从皮肤外植体培养的真皮成纤维细胞中测量， 小鼠为了产生实验小鼠，将Tsk和Tsk 2突变小鼠杂交 用携带各种胶原蛋白转基因构建体的转基因小鼠。一 Collal第一内含子在转录上调中的潜在作用 的Collal基因已被证明与Tsk和Tsk2突变（我们的 初步数据）以及在硬皮病真皮成纤维细胞中。具体目标 2 Collal第一内含子在Collal转录调控中的作用 基因和Tsk和Tsk 2纤维化皮肤表型的发展将是 测定对于这些实验，首先在Collal中进行靶向缺失， 将使用内含子。这个实验模型有一个独特的特点 允许测定由所述细胞产生的Co11a1 mRNA的水平， 缺失的和正常的等位基因。这种基因 该系统允许监测基因表达的背景下， 内源基因第二个实验方向涉及识别基因， 与胶原蛋白水平升高相关的真皮成纤维细胞 生产使用微阵列分析。概述的实验计划， 具体目标3包括开发试剂以分离特异性 从突变动物和正常动物培养的真皮成纤维细胞群 基于他们的胶原基因表达。这将通过使用 一种胶原启动子GFP报告基因转基因， 在分离自Tsk和Tsk 2的真皮成纤维细胞中表达升高 突变小鼠流式细胞术分析表达这种蛋白的真皮成纤维细胞 转基因将允许基于细胞群的表达水平分离细胞群。 胶原蛋白表达RNA将从高胶原蛋白和低胶原蛋白中提取 产生胶原蛋白的细胞群。这些RNA将用于 微阵列分析鉴定高胶原蛋白表达差异的基因 与低胶原蛋白产生细胞相比，反之亦然。是 预计在这个实验范例中鉴定的基因将允许 解剖参与发病的分子途径， 硬皮病，并可能导致治疗控制细胞外基质 新陈代谢.

DESCRIPTION (provided by applicant): This proposal will utilize two mouse mutations that are models for scleroderma, tight skin (Tsk) and tight skin 2 (Tsk2). Both mutations display excessive accumulation of collagen and other extracellular matrix components in the skin, a hallmark feature of the human disease. The long range of objective of the proposed research is to utilize these two mutations combined with several lines of transgenic mice as experimental tools to dissect molecular mechanisms of disease pathogenesis. Specific experiments are proposed for the identification of genes involved in the regulation of extracellular matrix synthesis in dermal fibroblasts. Two experimental strategies are planned and are encompassed in three specific aims. Specific aim 1 focuses on identifying cis-acting elements in the type I collagen gene required for the increased production of Collal mRNA in mutant dermal fibroblasts. Defining "fibrotic" specific elements will provide a basis for the identification of the transacting factors that interact with these DNA segments to increase Collagen gene expression. These elements will be defined by studying the expression of Collal CAT reporter transgenes bearing various segments of the 5' promoter region as well as specific deletions of the first intron. The expression of each transgene will be evaluated in skin samples isolated from Tsk, Tsk2 and normal mice. Also, transgene expression will be measured in dermal fibroblasts cultured from skin explants isolated from these mice. To generate experimental mice, Tsk and Tsk2 mutant mice will be crossed with transgenic mice bearing the various collagen transgene constructs. A potential role of the Collal first intron in the upregulation of transcription of the Collal gene has been shown with the Tsk and Tsk2 mutations (our preliminary data) as well as in scleroderma dermal fibroblasts. In specific aim 2 the role of the Collal first intron in regulating transcription of the Collal gene and the development of the Tsk and Tsk2 fibrotic skin phenotype will be determined. For these experiments a targeted deletion in the Collal first intron will be employed. This experimental model has a unique feature permitting the determination of the levels of Co11a1 mRNA produced by the deleted and normal allele in the same RNA preparation. Further this genetic system allows the monitoring of gene expression in the context of the endogenous gene. A second experimental direction involves identifying genes in dermal fibroblasts that are associated with elevated levels of collagen production employing micorarray analysis. The experimental plan outlined in specific aim 3 includes the development of reagents to isolate specific populations of dermal fibroblasts cultured from both mutant and normal animals based on their collagen gene expression. This will be accomplished by employing a collagen promoter GFP reporter transgene that has been documented to display elevated expression in dermal fibroblasts isolated from both Tsk and Tsk2 mutant mice. Flow cytometric analysis of dermal fibroblasts expressing this transgene will permit the isolation of cell populations based on their level of collagen expression. RNA's will be extracted from high collagen and low collagen producing cell populations. These RNA's will be utilized in a microarray analysis to identify genes differentially expressed in high collagen producing cells compared to low collagen producing cells and visa versa. It is anticipated that genes identified in this experimental paradigm will permit the dissection of molecular pathways that are involved with the onset of scleroderma and potentially lead to therapies to control extracellular matrix metabolism.