Expression of Multiple Transgenes from a Single Construct Using Viral 2A Peptides in Drosophila
Key findings
Comparison of 2A peptide separation efficiency in Drosophila S2 cells revealed P2A and T2A peptides achieved 96% and 88% cleavage efficiency respectively, significantly outperforming E2A (68%) and F2A (65%). Western blot quantification demonstrated that proteins before and after the P2A sequence were produced at equivalent levels (98% vs 93% normalized intensity, n=2), confirming ratiometric stoichiometry without translational bias or protein degradation.
PhiC31-mediated genomic integration of myrGFP-2A-dsRed-nls constructs into identical loci revealed that P2A and T2A peptides produced minimal perinuclear aggregates (<5 per cell) in larval salivary glands and muscles compared to E2A and F2A variants. Aggregate quantification showed E2A and F2A generated 2.3-fold more and 1.8-fold larger GFP/RFP-positive puncta, confirming tissue-independent superiority of P2A and T2A for polycistronic expression in vivo.
Co-expression of tdTomato-P2A-GCaMP5G in larval motorneurons enabled frequency-dependent calcium imaging (5-50 Hz stimulation) with motion artifact correction in intact, contracting preparations. The stable tdTomato signal (minimal fluctuation) allowed normalization of GCaMP5G fluorescence changes, revealing synaptic activity through the cuticle at the neuromuscular junction that was undetectable without ratiometric correction due to movement-induced signal artifacts.