TSK-2: A NEW ANIMAL MODEL FOR SCLERODERMA

系统性硬化症（SSc）是一种病因不明的严重疾病，其特征是皮肤和内脏中胶原蛋白和其他结缔组织成分的过度积累。建立动物模型对研究SSc的分子机制具有重要意义。我们最近开始了这样一个模型的研究，紧肤2或Tsk2小鼠。在之前的资助期间，我们最终证明了Tsk2是一种不同于Tsk1的突变；我们已经在1号染色体的近端臂上定位了突变；我们已经将突变发生的间隔从50厘米缩小到2.1厘米。我们已经证明，Tsk2小鼠显示出真皮的显著增厚和真皮胶原蛋白的过度积累。我们发现胶原蛋白合成和I、III、V和VI型胶原mRNA水平在Tsk2小鼠皮肤或真皮成纤维细胞中显著升高。I型和III型胶原基因的表达升高是由于相应基因的转录增加。这些结果表明，Tsk2突变小鼠显示结缔组织异常，类似于SSc患者和Tsk1小鼠皮肤中的结缔组织异常。另外一种Tsk突变的发现将使我们能够寻求第二种途径来理解在分子水平上控制胶原基因表达的机制，并且应该大大增加我们最终找到有效治疗SSc的机会。这项更新申请的总体目标是鉴定Tsk2基因。为了实现这一目标，我们将追求以下具体目标：(1)进一步缩小已知Tsk2所在的1号染色体区域；继续对[(castaneus x C57BL/6-+/Tsk2)F1 x castaneus]小鼠亚种间回交的N2后代进行分型和定位；(2)继续筛选已知存在于目标区域或目标区域附近的筛选候选基因；(3)建立包含Tsk2的YAC和BAC序列，并用重组标记物建立精确的近端和远端边界；(4)通过CpG岛鉴定、外显子捕获和cDNA选择等方法鉴定各组内的编码区；(5)筛选和鉴定新的基因序列。这些研究将使我们能够鉴定和克隆Tsk2，并将突变特征作为理解其功能的前奏。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，并将为开发这种无法治愈的毁灭性疾病的可能治疗模式提供更合理的方法。

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.