Drosophila Eggshell Production: Identification of New Genes and Coordination by Pxt

Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochromome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.The Drosophila Gene Disruption Project is supported by NIH NIGMS R01GM067858 to HJB. Confocal microscopy at the NRI is supported by the Neurovisualization Core of the IDDRC, funded by NIH U54HD083092. We thank the Harvard Medical School Image Data Management Core for support with the OMERO platform and the Harvard Medical School Division of Immunology’s Flow Cytometry Facility for the use of their FACS equipment. We also thank Raghuvir Viswanatha, Justin Bosch, Denise Lanza and Jason Heaney for helpful discussions, Robert Levis for critical reading and editing of the manuscript and Tzumin Lee for fly stocks. Development of the tagged cell line resource at the Drosophila RNAi Screening Center (DRSC) was supported by NIH ORIP R24 OD019847 (PI: NP, CoPI: A Simcox, Co-I: SEM). Additional relevant support for the DRSC includes NIH NIGMS R01 GM084947 and P41 GM132087 (PI: NP, Co-I: SEM). NP, ACS, and HB are investigators of Howard Hughes Medical Institute.Peer reviewe