Fig. 5
- ID
- ZDB-FIG-200924-25
- Publication
- Burgess et al., 2020 - An optimized QF-binary expression system for use in zebrafish
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Tagging endogenous loci with QFGal4. (A) Schematic showing the pGTag:QFGal4 gene tag construct. The P2A:QFGal4:SV40 expression cassette is flanked by left and right homology arms (LHA and RHA, respectively). In addition, the vector contains universal gRNA (UgRNA) target sequences, that can be cut in vivo by coinjection with Cas9 and UgRNA (modified from (Wierson et al., 2020)). (B) Confocal fluorescence micrographs showing GFPNLS expression in sox2-QFGal4 knock-in tagged cells. Tg(QUAS5x:GFPNLS)hsc143 transgenic lines were injected with pGTag:QFGal4, sox2 TALEN mRNA, as well as UgRNA and Cas9 protein to cut the pGTag vector in vivo. Successful QFGal4 integration at the sox2 locus was visualized by Tg(QUAS5x:GFPNLS)hsc143 reporter activity. Mosaic GFPNLS expression was visible throughout the spinal cord and retina in 3 dpf larvae (top two panels). GFPNLS labeling was visible in the retina and brain (lower left panel), hindbrain (middle lower panel) and spinal cord (lower right panel). (C) Fluorescence micrographs showing GFPNLS expression in ubiquitin C (ubc) expressing cells tagged with QFGal4. Tg(QUAS5x:GFPNLS)hsc143 transgenic lines were injected with pGTag:QFGal4, ubc gRNA, UgRNA and Cas9 protein. Epifluorescence micrographs show mosaic GFPNLS expression visible throughout the larval body, consistent with broad integration of QFGal4 at the ubc locus (3 dpf, top left panel and 5 dpf, top right panel). Confocal micrographs showing GFPNLS expression in a range of cells, including brain and retina (lower left panel), trunk (lower middle panel) and spinal cord, muscle, epidermal cells (lower right panel). |
Reprinted from Developmental Biology, 465(2), Burgess, J., Burrows, J.T., Sadhak, R., Chiang, S., Weiss, A., D'Amata, C., Molinaro, A.M., Zhu, S., Long, M., Hu, C., Krause, H.M., Pearson, B.J., An optimized QF-binary expression system for use in zebrafish, 144-156, Copyright (2020) with permission from Elsevier. Full text @ Dev. Biol.