RiboNucleic Acid interference
RNAi
RiboNucleic Acid interference (RNAi) is a process of reducing or silencing a gene’s expression through double-stranded RNA (dsRNA)-mediated destruction of the target mRNA. Within only a few years, RNAi has become a highly useful molecular biology tool to elucidate gene function in a wide range of eukaryotic organisms. In the model nematode species Caenorhabditis elegans, RNAi was used to screen 86% of the annotated genes in the worm’s genome (Kamath et al, 2003; Nature 421:231). While only10% of these gene knockdowns produced an observable phenotype, two thirds of them had no previously known mutant phenotype. In Drosophila melanogaster, RNAi has been used to study the functions of large numbers of genes in cell culture (Lum et al., 2003, Science 299:2039; Kiger et al., 2003, J Biol 2: 27) to identify new genes involved in cell growth, proliferation, and morphology. I propose to develop high throughput RNAi for use in intact Drosophila melanogaster insects, to identify the role of genes important to reproductive physiology and germ-line development. By targeting intact insects rather than individual cells in culture, we can identity the role of genes relevant to multicellular development. High throughput dsRNA expression vectors and transposon-based transformation vectors will be used to either in vitro transcribe dsRNAs or genetically transform insects with all targeted Drosophila genes. The in vitro transcribed dsRNAs can be delivered directly to insect embryos or various life stages (using various high throughput delivery systems such as biolistics or feeding) for transient RNAi bioassays. For those genes that fail to produce obvious RNAi phenotypes using transient gene silencing, embryos can be transformed with inducible RNAi expression cassettes, to examine the impact of a silenced gene on the animal’s phenotype at various stages of development. Many of the molecular biology steps in the cloning and in vitro production of dsRNA and even insect bioassays are amenable to the use of robotics for rapid technology development. Given the ease with which dsRNA can be delivered to cells in culture, robotic approaches to screening for the function of mammalian germ line genes is also appropriate. [What will be of importance in these screens is the choice of assay to validate function of genes involved in germ line development
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