TAMOXIFEN-INDUCED KNOCK-OUT OF A RETINAL GENE

这项建议旨在提供在分子生物学、生物化学和电生理学方面的多样化和广泛的研究经验，使申请者能够发展成为一名成熟的科学家和临床医生，具有为学术眼科和基础科学研究做出贡献的突出潜力。培训的所有组成部分都包含在加州大学洛杉矶分校EyeSTAR项目的课程中。该研究项目将专注于利用可诱导的基因敲除系统，在编码cGMP-磷酸二酯酶(PDEgamma)的伽马亚单位的基因中产生组织和时间特异性的修饰。PDEgamma的整个编码区两侧将由34个碱基对的Cre重组酶识别位点(LoxP)通过同源重组引入到基因座中。经过种系传递后，携带修饰等位基因的小鼠Pdes lox1/Pdes tm1将在PDEβ亚基(Pdeb)基因组位置的控制下与表达Cre重组酶/类固醇激素受体融合蛋白的小鼠杂交。Cre重组酶/类固醇激素受体融合蛋白的使用将允许外源应用类固醇来调节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是至关重要的。拟议中的研究将开发工具，简化在特定细胞和特定时间从基因组中删除基因的过程。了解Pdes lox1/Pdes tm1小鼠中光感受器信号和退化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。黛博拉·法伯博士因其对视网膜退行性变，特别是视网膜色素变性的生化和分子生物学研究而在全国范围内获得认可。法伯博士将担任国际私学博士后研究的导师。此外，加州大学洛杉矶分校拥有一批杰出的教职员工，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计划还包括三年的眼科临床培训，从而获得认证和专业委员会资格。这项为期三年的高强度实习计划提供了丰富的眼科问题诊断和治疗的临床经验，以及包括讲座、专题研讨会和出席临床研究会议在内的许多教学活动。

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.