Development of Novel Tools for Gene Targeting in Smooth Muscle

平滑肌基因靶向新工具的开发

• 批准号: 8328706
• 负责人: Rosalyn M Adam
• 金额: $ 26.1万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-06 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8328706
• 关键词: Affect; Alleles; Asthma; Automobile Driving; Biology; Biomedical Research; Bladder; Brain; Computer Simulation; Contractile Proteins; Data; Databases; Development; Differentiation Antigens; Discipline; Disease; Elements; Functional disorder; Gene Targeting; Genes; Genetic; Genetic Models; Genetic Recombination; Genomics; Healthcare; Heart; Homeostasis; Human; Hypertrophy; In Situ Hybridization; In Vitro; Informatics; Intestinal Obstruction; Investigation; Knock-in Mouse; Knock-out; Link; Mediating; Modeling; Mus; Muscle function; Myocardium; Myopathy; Myosin Heavy Chains; Myosin Light Chain Kinase; Organ; Organ Specificity; Pattern; Phenotype; Physiology; Play; Proteins; Purinoceptor; Reagent; Relative (related person); Reporting; Research; Role; Running; SM 22 muscle protein; Site; Smooth Muscle; Smooth Muscle Myosins; Specificity; Technology; Testing; Time; Tissues; Transcript; Transgenic Mice; Transgenic Organisms; Validation; Viscera; Visceral; base; body system; calponin; cell type; cohort; economic cost; environmental change; homologous recombination; in vivo; innovation; insight; motility disorder; novel; promoter; recombinase; research study; response; tool; translational study; unpublished works

DESCRIPTION (provided by applicant): Diseases characterized by aberrant smooth muscle (SM) function, such as bladder over activity, asthma, and motility disorders of the gut, affect tens of millions of people in the US each year. The economic cost of treating such conditions is substantial and runs into the tens of billions of dollars annually. Despite this significant healthcare burden, research in the field of SM biology, especially visceral SM, has lagged behind that in other disciplines. This results in part from the paucity of genetic models that are the mainstay of modern biomedical research. In this revised application we propose an innovative strategy for the identification and verification of candidate drivers for Cre recombinase expression in visceral SM. We believe that successful demonstration of selective gene targeting in SM, in an organ-specific manner, would represent a major breakthrough in the field of SM biology and would provide new opportunities for mechanistic and translational investigation of SM pathophysiology. Although SM-specific targeting of Cre has been reported, such models display Cre-mediated recombination in essentially all smooth muscle-containing tissues, and do not allow for organ-specific gene targeting. Furthermore, current strategies typically rely on promoters encoding SM contractile proteins such as SM-MHC and SM221 to achieve SM-specific targeting of Cre. However, this requires SM differentiation to have occurred before such promoters are active. To circumvent these limitations, we propose two complementary approaches. In Aim 1, we will exploit recent unpublished data from our group showing that expression of the purinergic receptor P2rx1 is highly restricted to bladder SM, to generate P2rx1-Cre knock-in and transgenic mouse lines and determine their utility for organ-selective, SM-specific gene targeting. In Aim 2, we will screen novel, candidate promoters, predicted from informatics analysis to be enriched in visceral SM, for their cell type- and organ-specificity, and their ability to drive Cre expression in an organ-specific, SM-specific manner. At the end of the 2-year project period, we expect to have developed a completely novel suite of SM- specific gene-targeting reagents that will provide, for the first time, an opportunity to knock out (or knock in) genes implicated in SM dysfunction in an organ-specific manner. We believe these additions to the genetic 'tool-kit' will greatly facilitate advancements in our understanding of SM biology.

描述（由申请人提供）： 以平滑肌 (SM) 功能异常为特征的疾病，例如膀胱过度活动、哮喘和肠道动力障碍，每年影响数千万美国人。治疗此类疾病的经济成本巨大，每年高达数百亿美元。尽管存在巨大的医疗负担，但 SM 生物学领域，特别是内脏 SM 的研究仍然落后于其他学科。造成这种情况的部分原因是缺乏作为现代生物医学研究支柱的遗传模型。 在此修订后的应用中，我们提出了一种创新策略，用于识别和验证内脏 SM 中 Cre 重组酶表达的候选驱动程序。我们相信，以器官特异性方式成功演示 SM 选择性基因靶向，将代表 SM 生物学领域的重大突破，并将为 SM 病理生理学的机制和转化研究提供新的机会。 尽管已经报道了 SM 特异性靶向 Cre，但此类模型在基本上所有含有平滑肌的组织中都显示了 Cre 介导的重组，并且不允许进行器官特异性基因靶向。此外，目前的策略通常依赖于编码 SM 收缩蛋白（例如 SM-MHC 和 SM221）的启动子来实现 SM 特异性靶向 Cre。然而，这需要 SM 分化在此类启动子活跃之前发生。为了规避这些限制，我们提出了两种互补的方法。在目标 1 中，我们将利用我们小组最近未发表的数据（显示嘌呤能受体 P2rx1 的表达高度限制于膀胱 SM）来生成 P2rx1-Cre 敲入和转基因小鼠系，并确定它们在器官选择性、SM 特异性基因靶向中的效用。在目标 2 中，我们将筛选新的候选启动子，根据信息学分析预测它们在内脏 SM 中富集，其细胞类型和器官特异性以及它们以器官特异性、SM 特异性方式驱动 Cre 表达的能力。 在两年的项目期结束时，我们预计将开发出一套全新的 SM 特异性基因靶向试剂，这将首次提供以器官特异性方式敲除（或敲入）与 SM 功能障碍有关的基因的机会。我们相信遗传“工具包”中的这些补充将极大地促进我们对 SM 生物学的理解的进步。