RESOLVING COMPLEX SYSTEMIC ENDOGENOUS EXPRESSION PATTERNS INTO SUBCELLULAR HIGH-RESOLUTION LOCALIZATION

将复杂的系统内源表达模式解析为亚细胞高分辨率定位

基本信息

批准号：
9164427
负责人：
Koen Jozef Theo Venken
金额：
$ 23.78万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9164427
关键词：
Address Affinity Chromatography Animal Model Applied Research Biochemical Biomedical Research Brain Cells Cellular Morphology Chimeric Proteins Collection Communities Complex DNA Transposable Elements DNA cassette Development Developmental Process Documentation Drosophila genus Drosophila melanogaster Drug-sensitive Ectopic Expression Embryo Event Exons Gene Expression Gene Expression Regulation Gene Fusion Gene Targeting Genes Genetic Genetic Transcription Genome Genomics Goals Image Imaging Device Individual Injection of therapeutic agent Knock-in Knock-out Label Libraries Life Light Location Maps Mediating Methods Microscopic Microscopy Modification Morphologic artifacts Mutate Nervous system structure Neuraxis Organism Pattern Phenotype Physiological Physiological Processes Protein Analysis Proteins Proteomics RNA RNA Splicing Reagent Regulation Research Resolution Resources Scientist Site System Technology Testing Tissues Transgenic Organisms Validation Whole Organism Work animal tissue fly gene function gene product genetic analysis genetic manipulation in vivo integration site mutant protein expression protein function public health relevance recombinase recombinase-mediated cassette exchange site-specific integration spectrograph tool

项目摘要

Project Summary The combination of phenotypic, genomic and proteomic analyses have readily advanced the determination of gene function in genetically tractable organisms such as Drosophila melanogaster. However, genetic analysis of complex physiological and developmental processes can be obscured by several factors including the delay between when a protein's biochemical function is lost and when a phenotype becomes apparent. Detailed imaging of a protein's endogenous expression is essential to provide a complete description of its function during the dynamics of developmental, physiological and even pathological events. On one hand, microscopic methods to examine expression in vivo generally provide a static view of expression. On the other hand, ectopic expression of a gene fused with a fluorescent marker can have artifacts from mis-expression. What scientists need is an easy method to generate an endogenously tagged protein, ideally one that can resolve expression to individual cells by simple genetic manipulation. This proposal develops such a method and validates it using Drosophila where the tagged gene can be placed in different mutant backgrounds to examine dynamic differences in protein expression. The authors use flies that have transposable elements inserted at defined genetic loci. The insertions are used as target sites to stably insert artificial exons encoding one of several fluorescent proteins that readily create a tagged gene fusion through RNA splicing. An alternative tag makes the gene's RNA product traceable rather than its protein product. A third tag facilitates the biochemical purification of a protein under its native expression conditions. Additionally, the expression of the tagged fusion protein in individual flies is subsequently narrowed down from the whole organism to clusters of cells or even individual cells. This refined expression is derived by the experimental control of both the tissue-specific and temporal regulation of recombinases that target sites flanking the artificial exons encoding the tags. The recombinases control a switch that regulates observable gene expression. This switch can be conditionally turned “ON” and “OFF” with FLP and Cre recombinases. Finally, reagents will be generated that will facilitate exchange of switchable reagents through simple crosses, making the method accessible to any fly geneticist. In conclusion, this proposal aims to develop a collection of over 100 endogenously tagged genes, 18 transgenic stocks expressing regulated recombinases and 24 transgenic stocks that permit the targeting of any gene in the fly with the proposed panel of tags. These reagents will be made freely available to the research community. While developed for flies, this method can readily be extended to other organisms.

项目概要表型、基因组和蛋白质组分析的结合很容易推进了确定然而，遗传易驯化的生物体（如果蝇）的基因功能。对复杂生理和发育过程的分析可能会因多种因素而变得模糊，包括蛋白质的生化功能丧失和表型变得明显之间的延迟。蛋白质内源表达的详细成像对于提供其完整描述至关重要一方面，在发育、生理甚至病理事件的动态过程中发挥作用。检查体内表达的显微镜方法通常提供表达的静态视图。另一方面，与荧光标记融合的基因的异位表达可能会因错误表达而产生假象。科学家需要的是一种简单的方法来生成内源标记蛋白，最好是能够该提案开发了这样一种方法，通过简单的基因操作来解决单个细胞的表达问题。并使用果蝇对其进行验证，其中标记的基因可以放置在不同的突变体背景中作者使用具有转座元件的果蝇检查蛋白质表达的动态差异。插入在确定的基因位点，插入物被用作稳定插入人工外显子编码的目标位点。几种荧光蛋白之一，可通过 RNA 剪接轻松创建标记基因融合体。替代标签使基因的 RNA 产品而不是其蛋白质产品可追溯。在天然表达条件下对蛋白质进行生化纯化。随后，单个果蝇中标记的融合蛋白从整个生物体缩小到簇这种精细的表达是通过对细胞甚至单个细胞的实验控制得出的。针对人工外显子编码侧翼位点的重组酶的组织特异性和时间调节重组酶控制调节可观察的基因表达的开关。使用 FLP 和 Cre 重组酶有条件地“打开”和“关闭”，最终将生成能够满足以下条件的试剂。将通过简单的杂交促进可切换试剂的交换，使任何苍蝇都可以使用该方法总之，该提案旨在开发 100 多个内源标记基因的集合， 18 个表达受调控重组酶的转基因原种和 24 个允许靶向带有建议的标签组的苍蝇中的任何基因将免费提供给虽然是为苍蝇开发的，但这种方法可以很容易地扩展到其他生物体。