The ultimate goal of a genome project must be to understand what the genes are doing, not merely identify their existence. We are yet some distance from achieving that goal in Drosophila. The availability of a gene targeting method is but the first step towards that goal. Ultimately, it would be desirable to have a knockout mutation for every gene in the fly to be able to elucidate the function of each of those 13,600 genes. The targeting method that has been developed is vastly more efficient and effective than prior methods that have been developed for flies, but the method is in its infancy. Many parameters of the system are uncharacterized, and it is currently a lengthy procedure, requiring approximately 4-6 months from design of the targeting construct to the production of a knockout mutation. The proposed work is designed to increase our knowledge of the parameters that affect the efficiency of the method, leading to more efficient construct design and manipulation of the flies. A variety of methods to make gene knockouts will be tested and characterized to find those that are efficient and generally suitable for introducing mutations into specific genes. Experiments will also be undertaken to develop methods to speed up the process of making mutations, by weeks or perhaps months. The result of this work will provide a guide to the most efficient route towards producing specific alterations in the Drosophila genome. Moreover, the results will determine whether it becomes feasible to contemplate a large-scale knockout project in Drosophila, and what the time scale of such a project will be. Finally, the availability of efficient methods for gene targeting in Drosophila will aid the use of Drosophila as a model for understanding the biology of human diseases.
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