Localization of transgenes for Drosophila models of myotonic dystrophy type 1
Authors: Andrea Waltrip; Noah Smith; Ginny Morriss
Affiliation: University of Mary Washington, Fredericksburg, VA
Keywords: d. trinucleotide repeat expansion; z. other (Transgene localization)
Myotonic Dystrophy Type 1, DM1, is a multi-systemic disorder that results from expansion of CTG repeats in the DMPK gene in humans. Drosophila melanogaster has been established as a model organism for the study DM1, by the construct of multiple transgenic DM1 lines containing different numbers of CTG repeats (60, 250, and 480), expressed using the GAL4/UAS system. Expression of long-repeat transgenes ((CTG)250 and i(CTG)480) has been shown to produce the phenotypes consistent with DM1, relative to control lines (i(CTG)60). The precise chromosomal location of insertion of the transgenes has not been reported. We are using both classical genetic and molecular approaches to localize CTG-repeat transgene insertion in the genome. To narrow down location to a specific chromosome, genetic crosses using GAL4 drivers on different chromosomes are being used to drive expression of repeat expansions to assess phenotypic ratios of eye color traits, specific to the transgenes, and flight capability, which has been shown to be defective in DM1 flies. We expect to see different phenotypic ratios in the F2 progeny from crosses using drivers localized to different chromosomes. We will confirm our chromosome localization using fluorescent in situ hybridization (FISH) of polytene chromosome preparations, using probes specific for the transgene insertion. Chromosome specific probes will be used to verify the chromosomal location. Since the FISH will allow us to narrow down the location of transgene insertion to a more specific region of the chromosome, we can target that specific region to more precisely determine the insertion site using PCR and sequencing. Preliminary results from the genetic analysis suggest that the (CTG)250 transgene is likely localized to chromosome 2 and that i(CTG)480 and i(CTG)60 are not likely localized to the chromosomes 1 or 2. Knowing the location of the transgenes can allow for more practical mating schemes to study various aspects of myotonic dystrophy disease mechanisms, but can also provide crucial information for understanding the expression of the transgenes, which may be influenced by nearby regulatory elements in the genome.