Abstract
Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.
Highlights
Research using transgenic zebrafish lines has greatly contributed to our understanding of vertebrate biology
We made a Gal4-ERT line driven by a 2.3 kb promoter region from the keratin 5 gene that has been reported to express in the epidermis exclusively [28]
Treating animals with 1 or 2 mM 4-OHT again produced notably higher mCherry expression levels and no sign of toxic effects (Figure S2F–G). These results indicate that the Gal4ERT system can be used to tune transgene expression to a desired level by titrating the concentration of 4-OHT
Summary
Research using transgenic zebrafish lines has greatly contributed to our understanding of vertebrate biology. Transgenic zebrafish are used widely for both gain and loss of function experiments as well as a means to track specific cell populations. All such studies require careful consideration regarding the location, timing, and levels of transgene expression. Constitutive ubiquitous promoters generally produce high levels of transgene expression and generate robust phenotypes, they do not differentiate cell-type specificity or the timing of gene function. To overcome these limitations, tissue specific promoters are used to direct transgene expression to discrete cell lineages and tissue types. Temporal control of transgene expression in zebrafish is typically achieved using heat-shock sensitive promoters [1], small molecule-controlled inducible promoters can control the timing and tune levels of transgene expression [2]
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