Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish.
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.
Davison
Jon M
JM
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Akitake
Courtney M
CM
Goll
Mary G
MG
Rhee
Jerry M
JM
Gosse
Nathan
N
Baier
Herwig
H
Halpern
Marnie E
ME
Leach
Steven D
SD
Parsons
Michael J
MJ
D000818:Animals; D030801:Animals, Genetically Modified; D016923:Cell Death; D004251:DNA Transposable Elements; D004742:Enhancer Elements, Genetic; D029968:Escherichia coli Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009601:Nitroreductases / Q000235:genetics; D009928:Organ Specificity; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism*
D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C070633:Gal-VP16; D015534:Trans-Activators; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C116054:NfsB protein, E coli; D009601:Nitroreductases