A Novel Approach for Directing Transgene Expression in Drosophila: T2A-Gal4 In-Frame Fusion
Key findings
Developed a T2A-Gal4 in-frame fusion that co-expresses Gal4 with the endogenous gene by exploiting the T2A 'ribosomal skipping' peptide. The method requires explicit knowledge only of the gene's open reading frame and not its regulatory elements, producing Gal4 expression in cells that express the target gene.
Depending on the site of insertion, the endogenous translation product may be truncated or left functionally intact, so T2A-GIFF can be tuned to express Gal4 in all expressing cells or selectively for particular splice variants.
Compared with four other 2A candidates in SL2 cells, the T2A sequence showed the highest efficiency: mCD8-T2A-EGFP yielded cytosolic/nuclear EGFP (not membrane-tethered), and mCD8-T2A-Gal4 produced quantitative nuclear Gal4 and robust UAS-reporter expression. In transgenic flies, bursicon-neuron T2A constructs drove UAS-RedStinger while control constructs lacking T2A did not.