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

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.