Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish

Abstract

Prior studies with transgenic zebrafish confirmed the functionality of the transcription factor Gal4 to drive expression of other genes under the regulation of upstream activator sequences (UAS). However, widespread application of this powerful binary system has been limited, in part, by relatively inefficient techniques for establishing transgenic zebrafish and by the inadequacy of Gal4 to effect high levels of expression from UAS-regulated genes. We have used the Tol2 transposition system to distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome. The vector uses the potent, hybrid transcription factor Gal4-VP16 to activate expression from a UAS:eGFP reporter cassette. In a pilot screen, stable transgenic lines were established that express eGFP in reproducible patterns encompassing a wide variety of tissues, including the brain, spinal cord, retina, notochord, cranial skeleton and muscle, and can transactivate other UAS-regulated genes. We demonstrate the utility of this approach to track Gal4-VP16 expressing migratory cells in UAS:Kaede transgenic fish, and to induce tissue-specific cell death using a bacterial nitroreductase gene under UAS control. The Tol2-mediated gene/enhancer trapping system together with UAS transgenic lines provides valuable tools for regulated gene expression and for targeted labeling and ablation of specific cell types and tissues during early zebrafish development.

Adaptation of GAL4 activators for GAL4 enhancer trapping in zebrafish

“…Grabher and Wittbrodt (2004) employed the fulllength VP16 activation domain in their fusion construct as the original GAL4VP16 construct (Sadowski et al, 1988). However, the other studies that employed "GAL4VP16" in zebrafish (Davison et al, 2007;Inbal et al, 2006;Köster and Fraser, 2001;Sagasti et al, 2005;Sassa et al, 2007;Scott et al, 2007) actually used GAL4VP16 (Barlev et al, 1995) instead of the original GAL4VP16 construct (Sadowski et al, 1988), although this fact was not explicit in these reports (personal communication from Dr. Köster, who first used GAL4VP16 in zebrafish; Köster and Fraser, 2001). In GAL4VP16 , the truncated VP16 activation domain (aa 413-470) was fused to GAL4 DBD in place of the full activation domain (aa 413-490).…”

Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish

“…Recently, we reported a highly efficient transgenesis method and gene trap and enhancer trap methods by using the Tol2 transposon system (13,14). More recently, we and others have taken advantage of these methods to isolate fish lines expressing Gal4-VP16 in specific tissues (9,15). In the present study we aim to further develop methodologies in zebrafish that enable targeted expression of a desired gene in desired cells.…”

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

“…Importantly, one of the most common methodologies ensuring cell-specific expression of transgenes in zebrafish is the Gal4-UAS binary system (derived from yeast), in which the transcription of genes placed 3 0 of an upstream activating sequence (UAS) relies on the DNA binding of the Gal4 transcriptional activator . Gene-and enhancer-trap methods have been applied to establish a significant number of Gal4 transgenic lines (Davison et al 2007;Scott and Baier 2009;Kawakami et al 2010;Balciuniene et al 2013), several of which are neural-specific (Scott et al 2007;. Notably, in these lines the Gal4 open reading frame (ORF) is randomly integrated in the fish genome through Tol2-based transposition, and the insertion site is not mapped; therefore, the sequence of the promoter elements driving Gal4 expression is unknown.…”

“…The possible integration of exogenous genes into any given loci and the analysis of their function in the living animal have dramatically improved over the past few years with the development of genome editing technologies. Prior to this recent explosion in the field of knockin generation, conventional transgenic zebrafish lines were generated by Tol2-mediated transgenesis, which has successfully allowed the making of hundreds of new reporter lines essential to the study of particular gene functions in vivo (Davison et al 2007;Scott and Baier 2009;Kawakami et al 2010;Balciuniene et al 2013). Bacterial artificial chromosome-based transgenesis has been and still is one of the go-to methods for making reporter lines.…”