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

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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.