Author response: A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

Abstract

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

The neuropeptide Drosulfakinin regulates social isolation-induced aggression inDrosophila

“…For Dsk-and Dilp2-GAL4 expression analysis a fluorescent reporter (Etheredge et al, 2018) carrying pJFRC105-10XUAS-IVS-NLS-tdTomato in VK00040 (a gift from Barret D. Pfeiffer, Rubin lab, Janelia Research Campus) and pJFRC29-10XUAS-IVS-myr::GFP-p10 in attP40 (Pfeiffer et al, 2012) was used. For examining CCKLR-17D1 expression, a MiMIC reporter (Nagarkar-Jaiswal et al, 2015) (BL 61771;y[1] w[*] Mi{PT-GFSTF.2}CCKLR-17D1[MI03679-GFSTF.2]) was used. For aggression and qPCR assays, comparisons were made between equivalent genetic backgrounds.…”

Selective Filopodia Adhesion Ensures Robust Cell Matching in the Drosophila Heart

“…Further co-staining with Fas3 and Tin antibodies, we found that Fas3 is highly expressed in Tin-positive CBs (Figure 3A), while at a much lower level in Svp-positive CBs. Confirmed through co-staining in both WT and endogenous Ten-m::GFPtagged embryos (MiMIC-Ten-m::GFP) (Nagarkar-Jaiswal et al, 2015), Ten-m expression shows a complementary expression pattern to Fas3, with relatively higher expression in Svp-positive CBs than Tin-positive CBs (Figures 3B, S3D, and S3D 0 ).…”

Misregulation of Drosophila Sidestep Leads to Uncontrolled Wiring of the Adult Neuromuscular System and Severe Locomotion Defects

“…We visualised Side at this stage using the GFP-exon trap line Mi00149-GFSTF (Nagarkar-Jaiswal et al, 2015), hereafter called SideGFP Mi00149 , that fuses with the cytoplasmic domain of Side to GFP at amino acid position 918 and does not disrupt protein function (see section Materials and Methods, Supplementary Figure 3). SideGFP Mi00149 was predominantly expressed in central discs areas (leg disc proper), but overall, it did not seem to reflect any ordered cell arrangements (Figures 1A,E).…”

“…Since Side protein is no longer detectable in late embryos using immunostainings (Sink et al, 2001;Siebert et al, 2009), we wanted to cheque if it is re-expressed during metamorphosis or if it is functionally replaced by one of its paralogs. To examine its postembryonic expression, we took advantage of a fluorescent exon-trap line that fuses GFP with the cytoplasmic domain of endogenous Side (Nagarkar-Jaiswal et al, 2015). In leg imaginal discs, Side was already expressed in Zfh1-positive adult muscle precursors in late third instar larvae, when adult motor axons begin to grow out of the CNS.…”

“…), also called CD8-GFP-Sh (Zito et al, 1999), was used as a control strain. Side GFP exon trap line and all other GFP exon trap lines listed in Table 1 were obtained from a collection of MiMIC protein trap lines (Nagarkar-Jaiswal et al, 2015). In SideGFP Mi00149 (BL#60507), the Mi00149-GFSTF.1 element is inserted between exons 16 and 17 of the side locus and leads to a fusion of amino acid 918 in the cytoplasmic domain of Side to a protein linker followed by GFP (913-QPSLN-GGGGS...).…”