Expression of Multiple Transgenes from a Single Construct Using Viral 2A Peptides in Drosophila

Daniels et al. (2014). PLoS ONE DOI: 10.1371/journal.pone.0100637 Citations: 167

Drosophila lacked a tested toolkit for choosing among viral 2A peptides. New shuttle vectors compared four 2A peptides for separation efficiency in cells and flies. P2A gave 96 percent separation and enabled tdTomato-GCaMP5G calcium imaging at the NMJ.

Parts used

T2A Self-Cleaving Peptide P2A Self-Cleaving Peptide E2A Self-Cleaving Peptide F2A Self-Cleaving Peptide tdTomato T2A (Drosophila)

Key findings

In Drosophila S2 cells with the actin5c promoter driving myrGFP-2A-dsRed-nls, the P2A (porcine teschovirus-1) and T2A (Thosea asigna virus) peptides separate the upstream myristoylated GFP from the downstream nuclear dsRed more completely than the E2A (equine rhinitis A virus) and F2A (foot-and-mouth disease virus) peptides. Anti-GFP western blot separation efficiency is 96 ± 1% for P2A and 88 ± 3% for T2A versus 68 ± 3% for E2A and 65 ± 4% for F2A (mean ± SEM, n = 5; p < 0.001 for E2A or F2A versus P2A or T2A by one-way ANOVA; P2A and T2A do not differ significantly). In a GFP-P2A-GFP-dsRed-nls construct in S2 cells, the anti-GFP bands for the protein upstream and downstream of P2A have similar normalized intensities (98 ± 2 versus 93 ± 7, mean ± SEM, n = 2), so the downstream protein is not strongly under-produced in this small experiment.
In Drosophila larvae, myrGFP-2A-dsRed-nls transgenes carrying E2A, F2A, P2A or T2A were integrated by phiC31 into the same genomic attP site to avoid position effects. Under Gal4 control, E2A and F2A give more perinuclear GFP/RFP-positive puncta, which mark unseparated myrGFP-2A-dsRed-nls protein, than P2A and T2A in salivary gland cells (act5c-Gal4) and in body wall muscle (how24B-Gal4). In muscle, E2A and F2A give more and larger aggregates (qualitative comparison, N = 2 cells per genotype, no statistics given). Whole-fly western blots show far less unseparated protein than S2 cells, so the difference between peptides may depend on the myristoyl and nuclear localization signals used and has not been shown to generalize to other cell types or stages.
Co-expressing tdTomato and GCaMP5G from one UAS-tdTomato-P2A-GCaMP5G transgene (n-syb-Gal4, Drosophila larval motoneurons) permits ratiometric calcium imaging at the larval neuromuscular junction (muscle 13, MN13-Ib boutons). In a dissected third instar preparation with muscle contraction blocked, GCaMP5G fluorescence in the terminals rises with increasing nerve stimulation frequency, and the tdTomato signal is stable, so normalizing GCaMP5G to tdTomato changes the trace little. In an intact, contracting larva imaged through the cuticle, the tdTomato signal fluctuates with muscle movement, raw GCaMP5G signals are difficult to interpret, and normalizing to tdTomato separates calcium-dependent signals from movement artifacts.

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Abstract

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.