A Novel Approach for Directing Transgene Expression in Drosophila: T2A-Gal4 In-Frame Fusion

Diao & White (2011). Genetics DOI: 10.1534/genetics.111.136291 Citations: 140

Endogenous Gal4 drivers required known regulatory elements, often unavailable. This paper fused T2A-Gal4 into rickets via viral T2A skipping, co-translating with the native protein. The driver matched rk's pattern and restored wing expansion via a rescue transgene.

Parts used

T2A Self-Cleaving Peptide

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.

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Abstract

In Drosophila, the Gal4-UAS system permits a transgene to be expressed in the same pattern as a gene of interest by placing the Gal4 transcription factor under control of the gene's DNA regulatory elements. If these regulatory elements are not known, however, expression of Gal4 in the desired pattern may be difficult or impossible. To solve this problem, we have developed a method for co-expressing Gal4 with the endogenous gene by exploiting the "ribosomal skipping" mechanism of the viral T2A peptide. This method requires explicit knowledge only of the endogenous gene's open reading frame and not its regulatory elements.