TSK-2: A NEW ANIMAL MODEL FOR SCLERODERMA

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的过度积累。因此，建立一种研究SSc分子机制的动物模型将是非常有用的。我们最近开始了对这种模型的研究，即Tight Skin 2或Tsk 2小鼠。在之前的资助期间，我们已经最终证明Tsk 2是一种不同于Tsk 1的突变;我们已经将突变定位在染色体1的近端臂上;并且我们已经将突变的间隔从超过50 cM缩小到2.1 cM。我们已经表明，Tsk 2小鼠显示出明显的真皮增厚和真皮胶原蛋白的过度积累。我们发现，胶原蛋白的合成和I，III，V和VI型胶原mRNA水平显着升高，无论是TSK 2小鼠皮肤或真皮成纤维细胞。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，Tsk 2突变小鼠显示结缔组织异常，这类似于SSc患者和Tsk 1小鼠皮肤中存在的结缔组织异常。发现额外的Tsk突变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，并将大大增加我们最终找到有效治疗SSc的机会。本次更新申请的总体目标是鉴定Tsk 2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Tsk 2存在于1号染色体上的区域;继续对来自[（Mus castaneus x C57 BL/6-+/Tsk 2）F1 x Mus castaneus]小鼠;（3）建立包含Tsk 2的YAC和BAC重叠群，并用重组标记建立精确的染色体和远端边界;（4）通过识别CpG岛、外显子捕获和cDNA选择子来识别重叠群中的编码区;（5）筛选和鉴定新的基因序列。这些研究将使我们能够识别和克隆Tsk 2，并将其突变特征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理解Ssc过度胶原沉积特征的发病机制直接相关，并将提供一种更合理的方法来开发这种不可治愈和毁灭性疾病的可能治疗模式。

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.