TAMOXIFEN-INDUCED KNOCK-OUT OF A RETINAL GENE

他莫昔芬诱导的视网膜基因敲除

• 批准号: 6384247
• 负责人: Stephen H Tsang
• 金额: $ 7.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384247
• 关键词: animal genetic material tag; biological signal transduction; biotechnology; cyclic GMP; electrodes; electrophysiology; electroretinography; enzyme activity; gene expression; gene targeting; genetically modified animals; green fluorescent proteins; laboratory mouse; phosphodiesterases; recombinase; retina degeneration; serial analysis of gene expression; southern blotting; tamoxifen; transfection /expression vector; visual photoreceptor

This proposal is designed to provide a diverse and extensive research experience in molecular biology, biochemistry, and electrophysiology that will allow the applicant to develop into a mature scientist-clinician with an outstanding potential to contribute to both academic ophthalmology and basic science research. All components of training are incorporated in the curriculum of the EyeSTAR program based at UCLA. The research project will focus on the generation of tissue- and time- specific modifications in the gene encoding the gamma subunit of cGMP- phosphodiesterase (PDEgamma) by using an inducible knockout system. The entire coding region of the PDEgamma will be flanked by 34-base pair Cre-recombinase recognition sites (loxP) introduced into the gene locus by homologous recombination. After germline transmission, mice Pdeglox1/Pdegtm1 bearing the modified allele will be crossed with mice expressing the Cre-recombinase/steroid hormone receptor fusion protein under the control of the genomic locus of the beta subunit of PDE (Pdeb). The use of Cre-recombinase/steroid hormone receptor fusion protein will allow the regulation of Cre-recombinase activity with exogenously applied steroids. Gaining temporal and spatial control of gene expression is essential for the elucidation of gene function in the whole organism. The proposed studies will develop tools that will ease the deletion of genes from the genome in specific cells and at specific times. Understanding of the mechanisms controlling photoreceptor signaling and degeneration in Pdeglox1/Pdegtm1 mice may help to develop strategies for the prevention or slowing of human retinal dystrophies. Dr. Debora Farber is nationally recognized for her biochemical and molecular biological studies of retinal degenerations, in particular retinitis pigmentosa. Dr. Farber will serve as mentor for the PI'S postdoctoral studies. In addition, UCLA has an outstanding panel of faculty members many of whom have professional relationships with Dr. Farber and can serve as scientific consultants to the PI. The proposed project will be at the cutting edge of current research is an ideal area with which to begin a scientific career. The EyeSTAR program also incorporates three years of clinical training in ophthalmology leading to certification and specialty board eligibility. This intense three-year residency program providers a vast clinical experience in diagnosis and treatment of the complete scope of ophthalmologic problems as well as a number of didactic activities including lectures, special seminars, and attendance at clinical research meetings.

该提案旨在提供分子生物学、生物化学和电生理学方面多样化和广泛的研究经验，使申请人能够发展成为一名成熟的科学家临床医生，具有为学术眼科和基础科学研究做出贡献的杰出潜力。培训的所有组成部分均纳入加州大学洛杉矶分校 EyeSTAR 项目的课程中。该研究项目将重点关注通过使用诱导型敲除系统在编码 cGMP 磷酸二酯酶 (PDEgamma) γ 亚基的基因中产生组织和时间特异性修饰。 PDEgamma 的整个编码区两侧是通过同源重组引入基因座的 34 碱基对 Cre 重组酶识别位点 (loxP)。种系传播后，携带修饰等位基因的 Pdeglox1/Pdegtm1 小鼠将与在 PDE (Pdeb) β 亚基基因座控制下表达 Cre 重组酶/类固醇激素受体融合蛋白的小鼠杂交。 Cre重组酶/类固醇激素受体融合蛋白的使用将允许通过外源应用的类固醇来调节Cre重组酶活性。获得基因表达的时间和空间控制对于阐明整个生物体中的基因功能至关重要。拟议的研究将开发工具，以方便在特定细胞和特定时间从基因组中删除基因。了解控制 Pdeglox1/Pdegtm1 小鼠光感受器信号传导和变性的机制可能有助于制定预防或减缓人类视网膜营养不良的策略。 Debora Farber 博士因其对视网膜变性（特别是色素性视网膜炎）的生化和分子生物学研究而获得全国认可。 Farber 博士将担任 PI 博士后研究的导师。此外，加州大学洛杉矶分校拥有一支优秀的教职人员小组，其中许多人与法伯博士有专业关系，可以担任 PI 的科学顾问。拟议的项目将处于当前研究的前沿，是开始科学生涯的理想领域。 EyeSTAR 计划还包含三年的眼科临床培训，以获得认证和专业委员会资格。这个为期三年的密集住院医师计划提供了诊断和治疗整个眼科问题的丰富临床经验，以及许多教学活动，包括讲座、特别研讨会和参加临床研究会议。