Zebrafish plasmid injection gave too few transgene-expressing cells for transient use. This paper used Gal4-VP16/UAS on one vector to amplify and tissue-restrict transgene expression. 94% of cells coexpressed two linked transgenes across vertebrate cell types.
Köster and Fraser demonstrated that Gal4-VP16-mediated transgene expression significantly increased the number of detectable transgene-expressing cells in zebrafish embryos compared to conventional CMV- or EF-1α-driven constructs. The Gal4-VP16 fusion activator, derived from the yeast DNA-binding domain coupled to the HSV VP16 activation domain, raised expression levels sufficiently for confocal imaging at 3 dpf.
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Using tissue-specific regulatory elements to drive Gal4-VP16, the authors reliably restricted UAS-dependent transgene expression to muscle, notochordal, or neuronal tissues in zebrafish embryos. Activator and effector units were encoded on a single injection construct, eliminating the need for stable transgenic line generation while enabling spatially controlled ectopic expression.
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Gal4-VP16 drove simultaneous coexpression of two UAS-dependent transgenes from a single construct in virtually all transgene-expressing cells, enabling real-time in vivo tracing with a GFP fluorescent reporter. The system functioned effectively in Xenopus embryos, chicken, mouse, and human cultured cells, confirming broad applicability across vertebrate model organisms.
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