New tool for late stage visualization of Oskar protein in Drosophila melanogaster oocytes

My goal was to look for new factors involved in osk translational control by monitoring Osk::GFP expression and ‘knocking down’ the expression of candidate genes by using RNA interference (RNAi). An expansive collection of transgenic RNAi (TRiP) lines target a large fraction of all Drosophila genes. Knockdown is achieved by crossing a chosen TRiP line to a GAL4 driver (MAT3), which directs TRiP expression in the germline cells of the ovary.

To simplify the screen I recombined the MAT3 driver and osk::GFP transgene on to the same chromosome. This allowed me to test each TRiP line with a single cross, with the TRiP transgene from one parent and osk::GFP and MAT3 from the other parent. To make the recombinant I crossed MAT3 and osk::GFP flies, and used a PCR assay to test the progeny of individual candidates for presence of MAT3 and Osk::GFP. To validate the knockdown approach, I crossed the osk::GFP MAT3 recombinant TRiP lines of known regulators of osk mRNA to see if I obtained the same phenotypes as previously observed for mutants of these genes. It was found that these knockdowns show no change in expression of Oskar::GFP protein in TRiP lines of activators and increased expression in TRiP lines of repressors, except for that of hephaestus, which exhibited decreased expression. vasa, which should not have exhibited any change in Osk expression, also appears to have decreased expression. In the future, we plan to use this recombinant in crosses with other TRiP lines of known genes used in osk translational control such as tudor, orb, and bruno. The knockdown of hephaestus, vasa, and aubergine should be reperformed in order to ensure that the results are accurate. In addition, this recombinant can be used in screens to find genes involved in osk expression during late stage oogenesis so that more detailed mechanisms of control in posterior development can be described.