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中胶原和其他结缔组织成分过度堆积。用动物模型来研究SSc的分子机制将是非常有用的。我们最近开始了对这种模型的研究，紧凑型皮肤2或Tsk2小鼠。在之前的资助期间，我们已经确凿地证明了Tsk2是一个不同于Tsk1的突变；我们已经将突变定位在染色体L的近臂上；我们已经将突变的间隔从50多cM缩小到了2.1 cM。我们发现Tsk2小鼠的真皮明显增厚，真皮胶原过度堆积。我们发现，无论是Tsk2小鼠皮肤还是真皮成纤维细胞，其胶原蛋白合成和I、III、V、VI型胶原mRNA水平均显著升高。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，这与SSC患者和Tsk1小鼠的皮肤中存在的异常相似。TSK基因突变的发现将使我们能够在分子水平上寻求另一种方法来了解控制胶原基因表达的机制，并将极大地增加我们最终找到治疗SSC的有效方法的机会。此次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号染色体上的区域；继续对[(Mus Castaneus x C57BL/6-+/Tsk2)F1 x Mus Castane us]小鼠亚种间回交的N2代进行分型和作图；(2)继续寻找已知位于感兴趣区域或其附近的筛选候选基因；(3)建立包含Tsk2的YAC和BAC重叠群，并利用重组标记建立精细的根和远端边界；(4)通过鉴定CpG岛、外显子捕获和cDNA选择来鉴定重叠群内的编码区；(5)筛选和鉴定新的基因序列。这些研究将使我们能够识别和克隆Tsk2，并将其表征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过度胶原沉积的发病机制直接相关，并将为开发这种无法治愈和毁灭性疾病的可能治疗模式提供更合理的途径。

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.