Synthetic smooth muscle cell-selective promoters

合成平滑肌细胞选择性启动子

• 批准号: 6848778
• 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/6848778
• 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（由申请人提供）：我们研究的总体目标是产生和表征在泌尿生殖道不同组织中具有活性的合成平滑肌细胞选择性启动子。这些启动子将是有用的靶向表达的基因，这些组织，他们将在我们的研究中使用，以确定介导平滑肌细胞选择性表达的蛋白质的分子机制。在目标1中，telokin启动子将用于产生在泌尿生殖道和消化道的平滑肌细胞中选择性表达EGFP的转基因小鼠。这些转基因小鼠将用于通过新鲜解离细胞的荧光激活细胞分选从这些组织中分离纯平滑肌细胞群。这些小鼠将提供给其他研究人员，需要纯群体的胃肠道平滑肌细胞的研究，他们也将在我们的研究，以确定介导平滑肌细胞选择性表达的蛋白质在不同组织的泌尿生殖道的分子机制。在目的2中，将产生由telokin和SM 22 β启动子的片段组成的合成嵌合启动子，并分析其指导13-半乳糖苷酶表达至转基因小鼠的泌尿生殖道的选定组织内的平滑肌细胞的能力。初步数据显示，telokin AT/CArG元件可以选择性地增加膀胱平滑肌细胞中的转基因表达。这表明，在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差异调节结合到该区域的telokin启动子的共同因子的活性。为了鉴定目标3中描述的这些因子实验，我们将比较已知能够结合该序列的蛋白质的表达，并且还使用AT/CArG元件作为亲和探针来分离存在于从EGFP转基因小鼠的膀胱、子宫和肠道获得的纯平滑肌细胞群体的核提取物中的蛋白质。在目标4中，提出使用全局蛋白质组学方法来鉴定在泌尿生殖道的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分析机制，调节平滑肌细胞选择性基因表达的泌尿生殖道。