Synthetic smooth muscle cell-selective promoters

描述（由申请人提供）：我们研究的总体目标是生成和表征在泌尿生殖道不同组织中活跃的合成平滑肌细胞选择性启动子。这些启动子将有助于在这些组织中靶向表达基因，并将在我们的研究中用于确定介导平滑肌细胞选择性表达蛋白质的分子机制。在Aim 1中，将使用该基因启动子来产生转基因小鼠，使其在泌尿生殖系统和消化道的平滑肌细胞中选择性地表达EGFP。这些转基因小鼠将通过荧光激活细胞分选新分离的细胞，从这些组织中分离出纯种的平滑肌细胞。这些小鼠将提供给其他需要纯尿道平滑肌细胞群体进行研究的研究人员，它们也将参与我们的研究，以确定在生殖道不同组织中介导平滑肌细胞选择性表达蛋白质的分子机制。在目标2中，将生成由端粒蛋白片段和SM22 (z)启动子组成的合成嵌合启动子，并分析其在转基因小鼠生殖泌尿道选定组织内将13-半乳糖苷酶表达到平滑肌细胞的能力。初步数据显示，AT/CArG元件可选择性地增加膀胱平滑肌细胞的转基因表达。这表明生殖道组织中的平滑肌细胞要么表达不同的转录因子，要么以不同的方式调节与端粒启动子区域结合的共同因子的活性。为了确定Aim 3中描述的这些因素，我们将比较已知能够结合该序列的蛋白质的表达，并使用AT/CArG元件作为亲和探针，分离EGFP转基因小鼠膀胱、子宫和肠道中获得的纯种平滑肌细胞核提取物中的蛋白质。在目标4中，建议使用全局蛋白质组学方法来鉴定在泌尿生殖道不同平滑肌组织中差异表达或修饰的其他核蛋白。总之，这些研究将提供一个全面的机制分析调节平滑肌细胞选择性基因表达在泌尿生殖道。

DESCRIPTION (provided by applicant): The overall goal of our studies is to generate and characterize synthetic smooth muscle cell-selective promoters that are active in different tissues of the genitourinary tract. These promoters will be useful for targeting expression of genes to these tissues and they will be utilized in our studies to determine the molecular mechanisms mediating smooth muscle cell-selective expression of proteins. In Aim 1 the telokin promoter, will be used to generate transgenic mice that selectively express EGFP in smooth muscle cells of the genitourinary and digestive tracts. These transgenic mice will be used to isolate pure populations of smooth muscle cells from these tissues by fluorescence activated cell sorting of freshly dissociated cells. These mice will be made available to other investigators that require pure populations of GU-tract smooth muscle cells for their investigations, they will also be in our studies to determine the molecular mechanisms mediating smooth muscle cell-selective expression of proteins in different tissues of the genitourinary tract. In aim 2, synthetic chimeric promoters comprised of fragments of the telokin and SM22 (z promoters will be generated and analyzed for their ability to direct 13-galactosidase expression to smooth muscle cells within selected tissues of the genitourinary tract in transgenic mice. Preliminary data show that the telokin AT/CArG element can selectively increase transgene expression in bladder smooth muscle cells. This suggests that smooth muscle cells in each of the tissues of the genitourinary tract must express either distinct transcription factors or differentially regulate the activity of common factors that bind to this region of the telokin promoter. To identify these factors experiments described in Aim 3, we will compare the expression of proteins known to be able to bind this sequence and also use the AT/CArG element as a affinity probe to isolate proteins present in nuclear extracts of pure populations of smooth muscle cells obtained from bladder, uterus and gut of EGFP transgenic mice. In aim 4 it is proposed to use a global proteomic approach to identify additional nuclear proteins that are differentially expressed or modified in distinct smooth muscle tissues of the genitourinary tract. Together these studies will provide a comprehensive analysis of the mechanisms regulating smooth muscle cell-selective gene expression in the genitourinary tract.