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

Area of Science:

• Molecular Biology
• Genetics
• Neuroscience

Background:

• Co-expression of multiple transgenes is crucial for complex experimental designs.
• Ribosome-skipping 2A peptides offer a method for generating multiple proteins from a single mRNA.
• Efficient methods for polycistronic expression in Drosophila are needed.

Purpose of the Study:

• To develop and validate methods for 2A peptide-mediated polycistronic expression in Drosophila.
• To identify the most efficient viral 2A peptides for Drosophila expression systems.
• To demonstrate the utility of this technique for advanced imaging applications.

Main Methods:

• Tested various viral 2A peptides for expression efficiency in cultured Drosophila cells and in vivo.
• Utilized porcine teschovirus-1 (P2A) 2A peptide for co-expressing tdTomato and GCaMP5G.
• Employed ratiometric calcium imaging with motion correction in Drosophila larval motorneurons.

Main Results:

• Porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) 2A peptides demonstrated superior separation efficiency.
• Successful co-expression of fluorescent reporters and calcium indicators was achieved.
• High-resolution synaptic activity at the neuromuscular junction was recorded in vivo.

Conclusions:

• 2A peptide technology provides an effective strategy for polycistronic gene expression in Drosophila.
• P2A and T2A peptides are highly efficient for generating multiple protein products from a single transcript.
• This approach facilitates advanced functional studies, such as calcium imaging in intact preparations.