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Drosophila melanogaster has become the organism of choice for a wide variety of questions in developmental biology because of the availability of mutations in many developmentally important genes as well as the elegant genetic tools available to manipulate the genome. Saturation mutagenesis has been achieved for a myriad of developmental processes, and sophisticated approaches have been developed to study the cell and tissue specific expression or deletion of genes. Through the use of transposon insertional mutagenesis cloning of new genes has been greatly accelerated. However, when the Drosophila genome project is completed, there will be many open reading frames for which there will be no known mutation. As many as two-thirds of potential genes have no known mutations. The ability to target mutations to genes defined solely by DNA sequence will become an indispensable part of analyzing function. Homologous recombination between DNA sequences, mutagenized in vitro, and the chromosome homologue has provided both yeast and murine cell and developmental biologist the opportunity to produce "knock-out" (loss-of-function) and "knock-in" (gene modifications) mutations in both organisms with enormous benefit to understanding function of all parts of the genome. We propose to develop a system to accomplish homologous recombination in Drosophila melanogaster. We will develop germline stem cell cultures, determine the best manner to transfect DNA and select for transformation and homologous recombination, and re-introduce these mutated cells back into the germline. We have shown that germline stem cells in the bag of marbles tumorous ovaries can be grown in vitro and that they can be reintroduced into the female germ line. We will determine whether these cells can produce functional oocytes and what are the optimal conditions for both growth of these cells and for introducing DNA for homologous recombination. In addition, we will explore the use of wild-type ovarian stem cells or embryonic pole cells as other sources of cells in which to achieve homologous recombination. These procedures will provide the opportunity to test the function of potential genes for which we lack mutations.
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