Genetic analysis of the Drosophila slowpoke gene and development of a novel method of targeted downregulation of gene expression by Gal4-repressor.

摘要

The slowpoke gene encodes the alpha-subunit of a large conductance calcium-activated potassium channel that exists in a variety of excitable and non-excitable cells. The channels can be activated by membrane depolarization alone, intracellular Ca2+ alone, or synergistically by depolarization and Ca2+. Slowpoke channels contribute to the repolarizing current of action potentials in Drosophila neurons and muscles. The slowpoke mutants exhibit a prolonged repolarization of the action potential which brings about behavioral defects such as a decrease in ethanol sensitivity and defective locomotion. We have investigated the functions of the Drosophila slowpoke gene by employing distinctive strategies such as FLP-FRT mediated recombination and tissue-specific manipulation of gene expression. Through the application of these methods, we were able to collect new information about the phenotypic contribution of the gene. Our results differ from previous findings and raise interesting questions that need to be resolved to more fully understand slowpoke gene function. Results presented here demonstrate that (1) two slowpoke deletion lines we generated turned out to be embryonic lethal, in contrast to the viable null allele (slo 4) previously created by other researchers, (2) muscle-specific slowpoke expression contributes to locomotor behaviors such as climbing and flying, and (3) constitutive slowpoke knockdown delayed adult development in a gender-specific manner. In addition, we developed a Gal4-Repressor, that can be a novel genetic tool for loss-of-function studies in Drosophila. We report that the Gal4-repressor is capable of down-regulating gene expression from nearby endogenous promoters. The use of the Gal4-repressor could possibly become a tool in the forward genetic analysis of a variety of species, since the Gal4-UAS binary system has been used in other genetic model organisms.