Hostile takeover (Hto) system: frequently asked questions
What is Hto?
Hto is a genetic system for studying gene/protein function in the fruit fly, Drosophila melanogaster. It consists of a small transposon designed to both misexpress and tag an endogenous coding region, and a set of screening strategies and downstream analyses that can be performed using the Hto transposon.
What is the vector?
Hto is flanked by inverted repeats from the Minos transposon; see our intro page. Minos shows very little tendency to hop into certain loci (hotspots) or avoid certain loci (coldspots), unlike the more widely used P element vector. Likewise it will hop into any region of a gene, whereas the P element prefers the 5′ ends of genes (see Bellen et al. 2011). The Hto vector was originally placed in the genome using a P element, but the preferred starter chromosome for new screens has only Hto and no P sequence remaining.
What is the reading frame?
The construct is in frame 0. The ORF from mCherry RFP (red in the figure below) leads into a 4-aa linker (RPQA) which ends on a full codon, just before the gtaagt splice junction (orange). An in-frame splice requires that the target exon begins with a full codon. The frame +1 and +2 versions of Hto have not been made, but this could easily be done by modifying the NotI site at the end of the Hto exon.
Can Hto be used for early embryo or germ line expression?
We used the UAS/promoter fragment from the original EP element, so we expect little or no germline expression, but this was not tested. Drivers for the embryo such as engrailed-GAL4 work well.
What is the genetic marker for seeing Hto inserts?
Hto has no white+ marker gene, and so the lines are white-eyed. We left out white in order to keep the element small and minimally disruptive. Most lines have a distinctive GMR>Hto eye phenotype that we use to score for the construct in crosses. Hto can usually also be detected by crossing to GMR-GAL4 and scoring the presence of RFP under a fluorescence dissecting scope. Below is such an image featuring two sibs with GMR, one with [Hto]KCM and one without. RFP is usually scorable even if the eye is severely disrupted. However, a few lines do not make a detectable fusion protein even if they have a GAL4 phenotype.
How does Hto relate to standard Drosophila protein trapping?
Original GFP protein traps use a transposon with an exon carrying the GFP coding region; this exon can be spliced in between two existing exons of a target gene (A vs B in the figure below), to generate a tagged version of the target protein (C). See flytrap.med.yale.edu.
–In a standard protein trap, the target gene is expressed only under control of its own endogenous regulatory elements. Hto lines are instead expressed under investigator control using the GAL4/UAS system. We have found a few Hto lines, such as BRW, that are also enhancer traps and express in particular tissues without GAL4.
–Standard protein traps are meant to report the normal localization pattern of the target protein, and not intended to produce mutant phenotypes. (However, they may disrupt the target protein if the GFP segment interrupts a folded domain of the target protein.) On the other hand, Hto is intended to produce mutant phenotypes for downstream genetic analysis.
–Depending on the insertion site, an Hto insert may express a C-terminal subfragment of the target protein, whereas standard protein traps should not do this.
