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

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.

SUBMITTER: Daniels RW

PROVIDER: S-EPMC4063965 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Daniels Richard W RW Rossano Adam J AJ Macleod Gregory T GT Ganetzky Barry B

PloS one 20140619 6

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 vit ...[more]

PMID: 24945148

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Project description:IntroductionThe controlled expression of many genes, including G-protein coupled receptors (GPCRs), is important for delineating gene functions in complex model systems. Binary systems for inducible regulation of transgene expression are widely used in mice. One system is the tTA/TRE expression system, composed of a tetracycline-dependent DNA binding factor and a separate tetracycline operon. However, the requirement for two separate transgenes (one for each tTA or TRE component) makes this system less amenable to models requiring directed cell targeting, increases the risk of multiple transgene integration sites, and requires extensive screening for appropriately-functioning clones.MethodsWe developed a single, polycistronic tetracycline-inducible expression platform to control the expression of multiple cistrons in mammalian cells. This platform has three basic constructs: regulator, responder, and destination vectors. The modular platform is compatible with both the TetOff (tTA) and TetOn (rtTA) systems. The modular Gateway recombineering-compatible components facilitate rapidly generating vectors to genetically modify mammalian cells. We apply this system to use the elongation factor 1? (EF1?) promoter to drive doxycycline-regulated expression of both the fluorescent marker mCherry and an engineered Gs-coupled GPCR "Rs1" separated by a 2A ribosomal skip site.ResultsWe show that our combined expression construct drives expression of both the mCherry and Rs1 transgenes in a doxycycline-dependent manner. We successfully target the expression construct into the Rosa26 locus of mouse embryonic stem (ES) cells. Rs1 expression in mouse ES cells increases cAMP accumulation via both basal and ligand-induced Gs mechanisms and is associated with increased embryoid body size. Heterozygous mice carrying the Rs1 expression construct showed normal growth and weight, and developed small increases in bone formation that could be observed in the calvaria.ConclusionsOur results demonstrate the feasibility of a single-vector strategy that combines both the tTA and TRE tetracycline-regulated components for use in cells and mouse models. Although the EF1? promoter is useful for driving expression in pluripotent cells, a single copy of the EF1? promoter did not drive high levels of mCherry and Rs1 expression in the differentiated tissues of adult mice. These findings indicate that promoter selection is an important factor when developing transgene expression models.

Dataset Information

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

Publications

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

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