The Drosophila RNA-binding protein Rumpelstiltskin (Rump) is a tissue-specific antagonist of the gypsy insulator, but only in tissues outside of the CNS. Depletion of Rump improves both enhancer blocking and barrier activities of the gypsy insulator in non-CNS tissue, and insulator complex localization is altered specifically in non-CNS tissue of rump mutants. Rump coimmunoprecipitates with gypsy insulator proteins, and genome-wide profiling of Rump by ChIP-seq in non-CNS Kc167 cells revealed extensive overlap with a subset of gypsy insulator sites. In contrast, Rump does not associate with chromatin in CNS-derived BG3 cells. These results suggest that the presence of Rump may interfere with higher order insulator interactions. To answer this question we have carried out Chromosome Conformation Capture (3C) experiments in rump mutant flies and also in Kc167 Rump knockdown cells, exploring the well characterized endogenous gypsy insulator regions Homie and 1A-2 Su(Hw) binding sites. In order to identify additional insulator sites that may be regulated by Rump, we have also carried out genome-wide ChIP-seq analysis of insulator proteins in rump mutants. Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. We have described a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.
Showing the most recent 10 out of 17 publications
