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

• Published in: PloS one 2014-06, Vol.9 (6), p.e100637-e100637
• Main Authors: Daniels, Richard W, Rossano, Adam J, Macleod, Gregory T, Ganetzky, Barry
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Biology and Life Sciences; Calcium; Calcium imaging; Cloning; Coding; Cuticle; Deoxyribonucleic acid; DNA; Drosophila; Drosophila melanogaster - cytology; Drosophila melanogaster - metabolism; Enzymes; Fluorescence; Foot & mouth disease; Gene Expression; Genetic Engineering; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Insects; Laboratories; Larva - cytology; Larva - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Motor neurons; Motor Neurons - cytology; Motor Neurons - metabolism; mRNA; Peptides; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Physiology; Plasmids; Plasmids - chemistry; Plasmids - metabolism; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Red fluorescent protein; Research and Analysis Methods; RNA; Teschovirus - genetics; Teschovirus - metabolism; Transgenes; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0100637

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.