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.
Highlights
One of the most powerful techniques for characterizing gene function is to generate transgenic animals in which an epitope tag such as GFP has been fused to the gene at its normal genomic location (Ross-Macdonald et al, 1999; Morin et al, 2001; Skarnes et al, 2004)
We previously developed a flexible system for engineering the Drosophila genome using the Minos-Mediated Integration Cassette (MiMIC) transposable element
Its content can be replaced by Recombination-Mediated Cassette Exchange (RMCE) leading to the introduction of any desired DNA, such as an artificial exon that encodes a protein tag
Summary
One of the most powerful techniques for characterizing gene function is to generate transgenic animals in which an epitope tag such as GFP has been fused to the gene at its normal genomic location (Ross-Macdonald et al, 1999; Morin et al, 2001; Skarnes et al, 2004). Previous methods for generating protein trap alleles in Drosophila have allowed only about 800 genes to be successfully tagged (Kelso et al, 2004; Buszczak et al, 2007; Quinones-Coello et al, 2007; Aleksic et al, 2009; Lowe et al, 2014). MiMIC carries sequences that function as a gene and protein trap when inserted in the proper orientation in a coding intron.
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