A genetic toolkit for tagging intronic MiMIC containing genes

Abstract

Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.DOI: http://dx.doi.org/10.7554/eLife.08469.001

Differentiating Drosophila female germ cells initiate Polycomb silencing by altering PRC2 sampling

“…We used a previously described crossing strategy employing both FLP-mediated and phiC31-catalyzed site-specific recombination (Nagarkar-Jaiswal et al, 2015) to efficiently introduce the silencing reporter into 109 genomic sites that already contained a single MiMIC transposon located within chromatin predicted to be active or repressed (Fig. 1B,C).…”

Transsynaptic interactions between IgSF proteins DIP-α and Dpr10 are required for motor neuron targeting specificity inDrosophila

“…DIP-α is a cell surface protein, and analysis of its subcellular localization might provide insights into its function. To address where DIP-α localizes within MNISN-1s, we used a 'protein trap', DIP-α-GFP-DIP-α, constructed by using RMCE to insert GFP into the reading frame of the same MiMIC as for the T2A-GAL4 replacement (Nagarkar-Jaiswal et al, 2015a). If such protein traps are expressed well, they usually are transported to the subcellular compartments where the endogenous protein is located.…”

FlyBase at 25: looking to the future

“…Over the last decade, there has been an additional focus on other types of data, with high-throughput data projects featuring prominently. Recent years have seen the completion of Release 6 of the Drosophila melanogaster reference genome (4,5); a complete reannotation of the reference genome on the basis of RNA-Seq coverage data, RNA-Seq junction data, and transcription start site profiles from the modENCODE project (6–10); and the generation of large public collections of insertion mutants by the Gene Disruption Project (11–14). …”