TSK-2: A NEW ANIMAL MODEL FOR SCLERODERMA

TSK-2：硬皮病的新动物模型

基本信息

批准号：
6374985
负责人：
PAUL J CHRISTNER
金额：
$ 21.38万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-09 至 2005-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6374985
关键词：
CpG islands artificial chromosomes chromosome walking collagen disease /disorder model gene expression gene mutation genetic mapping genetic strain histopathology laboratory mouse model design /development molecular pathology recombinant DNA systemic scleroderma tissue /cell culture

项目摘要

Systemic sclerosis (SSc) is a serious disease of unknown cause, characterized by excessive accumulation of collagen and other connective tissue components in the skin and internal organs. An animal model to study the molecular mechanisms of SSc would be extremely useful. We have recently initiated studies of such a model, the Tight Skin 2 or the Tsk2 mouse. During the previous period of funding we have conclusively demonstrated that Tsk2 is a different mutation than Tsk1; we have located the mutation on the proximal arm of chromosome l; and we have narrowed the interval in which the mutation lies from over 50 cM to 2.1 cM. We have shown that the Tsk2 mouse displays marked thickening of the dermis and excessive accumulation of dermal collagen. We found that collagen protein synthesis and type I, III, V and VI collagen mRNA levels were markedly elevated in either Tsk2 mouse skin or dermal fibroblasts. The elevated expression of type I and III collagen genes was due to increased transcription of the corresponding genes. These results demonstrated that the Tsk2 mutant mouse displays connective tissue abnormalities, which resemble those present in the skin of both SSc patients and Tsk1 mice. The discovery of an additional Tsk mutation will allow us to pursue a second avenue of approach to understanding the mechanisms controlling collagen gene expression at the molecular level and should greatly increase our chances of eventually finding an effective treatment for SSc. The overall goal of this renewal application is to identify the Tsk2 gene. To accomplish this goal we will pursue the following specific aims: (1) To further narrow the region on chromosome 1 on which Tsk2 is known to reside by ;continuing to type and map the N2 offspring from the intersubspecific backcross of [(Mus castaneus x C57BL/6-+/Tsk2)F1 x Mus castaneus] mice; (2) to continue to identify screen candidate genes which are known to reside in or near the region of interest; (3) to establish YAC and BAC contigs encompassing Tsk2 and establish refined proxinal and distal boundaries with recombinant markers; (4) to identify coding regions within the contigs by means of identifying CpG islands, exon trapping and cDNA selecton; and (5) to screen and identify the novel gene sequences. These studies will allow us to identify and clone Tsk2 and to characterize the mutation as a prelude to understanding its function. It is expected that the knowledge gained from these studies will be of direct relevance to the understanding of the pathogenesis of the excessive collagen deposition characteristic of Ssc and will provide a more rational approach to develop possible modes of therapy for this incurable and devastating disease.

系统性硬化症（SSc）是一种原因不明的严重疾病，其特征是皮肤和内脏器官中胶原蛋白和其他结缔组织成分过度积累。研究 SSc 分子机制的动物模型将非常有用。我们最近开始了对这种模型 Tight Skin 2 或 Tsk2 小鼠的研究。在之前的资助期间，我们已经最终证明 Tsk2 是与 Tsk1 不同的突变；我们已将突变定位在染色体 l 的近端臂上；我们将突变的区间从50cM以上缩小到2.1cM。我们已经证明，Tsk2 小鼠的真皮显着增厚，真皮胶原蛋白过度积累。我们发现 Tsk2 小鼠皮肤或真皮成纤维细胞中胶原蛋白合成以及 I、III、V 和 VI 型胶原蛋白 mRNA 水平显着升高。 I 型和 III 型胶原蛋白基因表达升高是由于相应基因转录增加所致。这些结果表明，Tsk2 突变小鼠表现出结缔组织异常，类似于 SSc 患者和 Tsk1 小鼠皮肤中存在的异常。额外 Tsk 突变的发现将使我们能够寻求第二种方法来了解在分子水平上控制胶原蛋白基因表达的机制，并且应该大大增加我们最终找到有效治疗 SSc 的机会。该更新申请的总体目标是识别 Tsk2 基因。为了实现这一目标，我们将追求以下具体目标： (1) 进一步缩小 1 号染色体上已知 Tsk2 所在的区域；继续对 [(Mus castaneus x C57BL/6-+/Tsk2)F1 x Mus castaneus]小鼠； (2) 继续筛选已知位于感兴趣区域或其附近的候选基因； (3) 建立包含 Tsk2 的 YAC 和 BAC 重叠群，并使用重组标记建立精细的近端和远端边界； (4) 通过识别CpG岛、外显子捕获和cDNA选择来识别重叠群内的编码区； (5)筛选和鉴定新基因序列。这些研究将使我们能够识别和克隆 Tsk2，并表征突变，作为了解其功能的前奏。预计从这些研究中获得的知识将与了解 Ssc 过度胶原沉积特征的发病机制直接相关，并将提供更合理的方法来开发这种无法治愈的破坏性疾病的可能治疗模式。