TAMOXIFEN-INDUCED KNOCK-OUT OF A RETINAL GENE

本申请旨在为申请人提供在分子生物学、生物化学和电生理学方面丰富多样的研究经验，使申请人能够发展成为一名成熟的科学家-临床医生，在眼科学术和基础科学研究方面都有突出的潜力。培训的所有组成部分都纳入了加州大学洛杉矶分校EyeSTAR项目的课程。该研究项目将侧重于通过诱导敲除系统在编码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个碱基对的cre -重组酶识别位点（loxP）。将携带修饰等位基因的小鼠Pdeglox1/Pdegtm1在PDE β亚基基因组位点的控制下与表达cre -重组酶/类固醇激素受体融合蛋白的小鼠进行种系传代。使用外源性类固醇激素受体融合蛋白可以调节cre -重组酶的活性。获得基因表达的时间和空间控制对于阐明整个生物体的基因功能至关重要。拟议的研究将开发工具，以减轻特定细胞和特定时间基因组中基因的缺失。了解Pdeglox1/Pdegtm1小鼠光感受器信号传导和变性的控制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博士是全国公认的视网膜变性，特别是视网膜色素变性的生化和分子生物学研究。法伯博士将担任PI博士后研究的导师。此外，加州大学洛杉矶分校有一个杰出的教师小组，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。所提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计划还包括三年的眼科临床培训，以获得认证和专业委员会资格。这个为期三年的住院医师培训项目提供了丰富的眼科问题诊断和治疗的临床经验，以及一系列的教学活动，包括讲座、特别研讨会和参加临床研究会议。

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.