Synthetic smooth muscle cell-selective promoters

合成平滑肌细胞选择性启动子

• 批准号: 6701180
• 负责人: BRIAN Paul HERRING
• 金额: $ 28.6万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6701180
• 关键词: flow cytometry; genetic promoter element; genetically modified animals; green fluorescent proteins; intestines; laboratory mouse; mass spectrometry; muscle cells; muscle proteins; myosin light chain kinase; protein binding; protein purification; recombinant DNA; smooth muscle; synthetic nucleic acid; tissue /cell culture; transcription factor; two dimensional gel electrophoresis; urinary bladder; uterus

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.

描述(申请人提供)：我们研究的总体目标是产生和表征合成的平滑肌细胞选择性启动子，这些启动子活跃在泌尿生殖道的不同组织中。这些启动子将有助于基因在这些组织中的靶向表达，它们将被用于我们的研究，以确定介导平滑肌细胞选择性蛋白质表达的分子机制。在目标1中，将使用telokin启动子来产生转基因小鼠，这些转基因小鼠在生殖道和消化道的平滑肌细胞中选择性地表达EGFP。这些转基因小鼠将被用来通过对新鲜分离的细胞进行荧光激活的细胞分选，从这些组织中分离出纯的平滑肌细胞群。这些小鼠将提供给其他研究人员，这些研究人员需要纯粹的胃肠道平滑肌细胞群进行研究，它们也将在我们的研究中确定在生殖道不同组织中介导平滑肌细胞选择性蛋白质表达的分子机制。在目标2中，将产生由端粒蛋白和SM22(Z)启动子片段组成的合成嵌合启动子，并分析它们将13-半乳糖苷酶表达导向转基因小鼠生殖道选定组织内的平滑肌细胞的能力。初步研究表明，telokin AT/Carg元件可以选择性地增加膀胱平滑肌细胞的转基因表达。这表明，泌尿生殖道的每个组织中的平滑肌细胞必须表达不同的转录因子，或者以不同的方式调节与端粒蛋白启动子的这一区域结合的共同因子的活性。为了确定目标3中描述的这些因素实验，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元件作为亲和探针来分离从绿色荧光蛋白转基因小鼠的膀胱、子宫和肠道获得的纯系膜细胞核提取液中存在的蛋白质。在目标4中，建议使用全球蛋白质组学方法来鉴定在不同的泌尿生殖道平滑肌组织中差异表达或修饰的其他核蛋白。这些研究将全面分析泌尿生殖道中平滑肌细胞选择性基因表达的调控机制。