Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

Richard W Daniels,Gregory T Macleod,Adam J Rossano,Barry Ganetzky,Brian D Mccabe

doi:10.1371/journal.pone.0100637

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.

Highlights

Co-regulated expression of genes is important in many biological contexts and has great utility when introducing exogenous genes into model organisms
A Versatile Shuttle Vector for Cloning 2A Peptides As a first step to compare the properties of different 2A peptide sequences in Drosophila, we generated a vector that facilitates cloning of transgenes separated by the 2A sequence
After the desired construct has been generated in the pC5-Kan 2A shuttle vector, it can be subcloned into the pUAS-C5 expression vector using flanking rare restriction enzymes (Figure 1D)

Summary

Introduction

Co-regulated expression of genes is important in many biological contexts and has great utility when introducing exogenous genes into model organisms. An obvious application is to co-express a reporter gene with an effector to identify or ‘‘label’’ the cells manipulated by the effector gene. To achieve expression of multiple transgenes in cultured cells, separate plasmids, each with their own promoter, are co-transfected often in combination with a reporter plasmid at a lower concentration. Since the majority of cells that take up DNA will take up both plasmids, reporter-expressing cells are likely to express the co-transfected genes. While co-transfection with multiple plasmids is possible for in vitro cell transfection and even in vivo through electroporation, it is far from ideal for most in vivo applications as it can still result in genetically manipulated cells that are not marked by a reporter

Methods

Results

Conclusion