ZFIN Image: Bhatia et al., 2021, Figure 1

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Figure 1

Quantitative Spatial and Temporal Assessment of Regulatory element activity in Zebrafish (Q-STARZ) pipeline.

Diagramatic representation of the Q-STARZ pipeline. (A). Top: map of the landing pad vector. Bottom: scheme for generating stable transgenic ‘landing lines’. The landing pad vector is co-injected with Tol2 mRNA into one-cell stage wild-type embryos. Tol2-mediated recombination integrates the landing pad containing phiC31-attB sites flanking the tracking cis-regulatory element (CRE)-reporter cassette (SHH-SBE2, a CRE driving eGFP in the developing forebrain) at random locations in the zebrafish genome. F0 embryos showing mosaic eGFP expression are raised to adulthood. F1 embryos obtained by outcrossing F0 lines with wild-type zebrafish are screened for tracking CRE-driven reporter (eGFP) expression. Embryos where eGFP expression was only observed in the expected activity domain (forebrain) of the tracking CRE were raised to adulthood to establish stable ‘landing lines’. (B) Top: map of the dual-CRE dual-reporter vector. Bottom: scheme for replacing the tracking cassette in the landing line with the dual-CRE dual-reporter cassette containing the enhancers to be assayed for spatiotemporal activity. Assay vector and mRNA coding for phiC31 integrase are injected in one cell stage embryos derived from outcrossing F1 landing line with wild-type fish. Injected embryos were selected for loss of tracking CRE (SHH-SBE2)-driven eGFP fluorescence in forebrain and mosaic expression of both eGFP and mCherry resulting from the test CREs in the assay cassette. F0 transgenic lines were established from selected embryos and eGFP and mCherry expression imaged in F1 embryos derived from outcrossing these lines with wild-type fish. Signals from both reporters were observed in the activity domains of both CREs in F1 embryos bearing the assay constructs with ‘neutral’ sequence between the two CRE-reporter units (yellow signal seen in expressing tissues in the merge channel). However, eGFP and mCherry expression was restricted to tissues where the associated CREs are active upon inclusion of three copies of the chicken β-globin 5′HS4 (3XcHS4) insulator between the two CRE-reporter units.

Acknowledgments

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