Despite recent advances in chemotherapy, the human filarial infections remain important public health problems. Recently, the great advances have been made in our understanding of the repertoire of genes expressed in these parasites. However, the lack of methods of genetic manipulation and the paucity of parasite material has limited and will continue to limit studies of these organisms. Recently, it has been demonstrated that exogenous DNA sequences can be introduced into embryos of the filarial parasite Brugia malayi, employing biolistic methods and transfection with pantropic retroviral vectors. The overall goal of this proposal will be to employ these systems to overcome the limitations imposed by the lack of traditional genetics and the inability to obtain large amounts of parasite material.
The specific aims of this proposal are: 1. To employ the biolistic transient transfection system to identify cis acting factors important in controlling gene expression in B. malayi. Parameters to be explored will include identification of a minimal promoter for transient expression in B. malayi, the effect of 5' and 3' sequences important for message processing, and the effect of the addition of synthetic introns. 2. To develop pantropic retroviral vectors as a system for stable foreign gene expression in B. malayi embryos. 3. To employ pantropic retroviral vectors expressing oncogenes to create an immortalized cell line from B. malayi. 4. To determine if heterologous DNA present in transfected B. malayi embryos is stably inherited.
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