Quantitative spatial and temporal assessment of regulatory element activity in zebrafish
- MRC Human Genetics Unit, Institute of Genetics & Cancer, University of Edinburgh, United Kingdom
- Centre for Mammalian Synthetic Biology at the Institute of Quantitative Biology, Biochemistry, and Biotechnology, SynthSys, School of Biological Sciences, University of Edinburgh, United Kingdom
Figures
Figure 1 with 1 supplement
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 …
Figure 1—figure supplement 1
Diagrammatic representation of the gateway cloning strategy used for generating the landing pad (A) and dual-cis-regulatory element (CRE) dual-reporter vector (B).
The recombination sites used in each vector and other salient features are indicated on each vector map.
Figure 2 with 1 supplement
Characterisation of SHH-SBE2 landing line.
(A) Top: schematic of the design of the landing pad bearing SHH-SBE2 as the tracking cis-regulatory element (CRE). Below: CRE activity observed exclusively in the forebrain in F1 embryos with the …
Figure 2—figure supplement 1
Landing lines with tracking cis-regulatory element (CRE)-driven reporter gene expression influenced by site of integration.
Top: tracking CREs used in the landing line. Bottom: activities of the SOX9 (A) and PAX6-SIMO (B) CREs in the landing pad highly influenced by the site of integration indicated by eGFP expression in …
Figure 3 with 4 supplements
Quantitative assessment of tissue-specific enhancer activity and effect of insulation on crosstalk between cis-regulatory elements (CREs) in dual-CRE dual-reporter constructs.
Constructs carrying well-characterised CREs from the PAX6 locus (PAX6-7CE3, hindbrain enhancer, and PAX6-SIMO, lens enhancer). (A) Confocal images of 96hpf F1 embryos derived from founder lines …
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Figure 3—source data 1
Quantification data of eGFP and mCherry intensities in transgenic lines bearing the assay constructs described in Figure 3.
- https://cdn.elifesciences.org/articles/65601/elife-65601-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Assessment of tissue-specific cis-regulatory element (CRE) activity from dual-CRE dual-reporter constructs with neutral sequence between CREs.
Images shown for pool of F1 embryos (scale bar = 1000 μm) and individual embryos (scale bar = 100 μm) at different stages of embryonic development (24–96 hr post fertilisation) derived from founder …
Figure 3—figure supplement 2
Assessment of tissue-specific cis-regulatory element (CRE) activity from the dual-CRE dual-reporter replacement construct with one copy of insulator sequence.
Replacement constructs designed with previously well-characterised enhancers from PAX6 locus (PAX6-7CE3, hindbrain enhancer, and PAX6-SIMO, lens enhancer). Images shown for pool of F1 embryos (scale …
Figure 3—figure supplement 3
Assessment of tissue-specific cis-regulatory element (CRE) activity from the dual-CRE dual-reporter replacement construct with two copies of insulator sequence.
Replacement constructs designed with previously well-characterised CREs from PAX6 locus (PAX6-7CE3, hindbrain enhancer, and PAX6-SIMO, lens enhancer). Images shown for pool of F1 embryos (scale bar …
Figure 3—figure supplement 4
Assessment of tissue-specific cis-regulatory element (CRE) activity from the dual-CRE dual-reporter replacement construct with three copies of insulator sequence.
Replacement constructs designed with previously well-characterised CREs from PAX6 locus (PAX6-7CE3, hindbrain enhancer, and PAX6-SIMO, lens enhancer). Images shown for pool of F1 embryos (scale bar …
Figure 4 with 1 supplement
Live imaging of transgenic embryos to detect subtle differences in spatial and temporal enhancer activities.
(A) Top: schematic of assay construct with two enhancers from the mouse Shh locus active in developing forebrain (Shh-SBE2 and Shh-SBE4 driving eGFP and mCherry respectively). Below: snapshots of …
Figure 4—figure supplement 1
Genotyping assay for assessing successful integration of the replacement construct in the landing sites.
Top: position of the screening primers (SP1- 12) used for genotyping a replacement construct bearing SHH-SBE2 and SHH-SBE4 cis-regulatory elements (CREs). Bottom: PCR products obtained using the …
Figure 5
Quantitative assessment of altered CRE activity by disease-associated sequence variation.
Dye swap experiment with SHH-SBE2 enhancer wild-type Wt(C) allele and Mut(T) allele bearing a holoprosencepaly- associated mutation (A: Wt(C)-eGFP/ Mut(T)-mCherry; B: Wt(C)-mcherry/ …
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Figure 5—source data 1
Quantification data of eGFP and mCherry intensities in transgenic lines bearing the assay constructs described in Figure 5.
- https://cdn.elifesciences.org/articles/65601/elife-65601-fig5-data1-v1.xlsx
Videos
Video 1
Confocal imaging of 30 hr post fertilisation embryo derived from transgenic line bearing the Shh-SBE2gfp/3XcHS4/Shh-SBE4mCherry assay construct.
The distinct expression domains of SBE2 and SBE4 enhancers in the developing forebrain are seen in green and red, respectively, while the region where their activities overlap is depicted in yellow.
Video 2
Time-lapse video of embryo derived from transgenic line bearing the Shh-SBE2gfp/3XcHS4/Shh-SBE4mCherry assay construct.
Images were acquired from 54 to 69 hr post fertilisation, with a time interval of 1 hr. The distinct expression domains of SBE2 and SBE4 enhancers in the developing forebrain are seen in green and …
Video 3
Time-lapse video of embryo derived from transgenic line bearing the SHH-SBE2-Wtgfp/3XcHS4/SHH-SBE2-Mut-mCherry assay construct.
Images were acquired from 40 to 60 hr post fertilisation, with a time interval of 2 hr.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Commercial assay or kit | Gateway recombination cloning system | Invitrogen | 12535-019 | |
| Commercial assay or kit | Phusion high fidelity polymerase | NEB | M0530S | |
| Commercial assay or kit | TOPO TA Cloning Kit | Thermo Fischer Scientific | 451641 | |
| Commercial assay or kit | Plasmid purification columns | QIAGEN | 12123 | |
| Commercial assay or kit | PCR purification columns | QIAGEN | 28115 | |
| Commercial assay or kit | SP6 mMessage mMachine kit | Ambion | AM1340 | |
| Commercial assay or kit | DNeasy blood and tissue kit | QIAGEN | 69504 | |
| Commercial assay or kit | T4 ligase | NEB | M020S | |
| Recombinant DNA reagent | pCS2-TP (plasmid) | Bischof et al., 2007 | ||
| Recombinant DNA reagent | pcDNA3.1 phiC31 (plasmid) | Addgene | Plasmid #68310 | |
| Recombinant DNA reagent | NlaIII(enzyme) | NEB | R0125S | |
| Recombinant DNA reagent | BfaI(enzyme) | NEB | R0568S | |
| Recombinant DNA reagent | DpnII(enzyme) | NEB | R0543S | |
| Chemical compound, drug | PTU (1-phenyl2-thio-urea) | Sigma-Aldrich | S515388 | |
| Chemical compound, drug | Low-melting point (LMP) agarose | Sigma-Aldrich | A9414 | |
| Chemical compound, drug | Tricaine | Sigma-Aldrich | MS222 | |
| Software, algorithm | Imaris | Bitplane, Oxford Instruments | RRID:SCR_007370 | |
| Software, algorithm | Fiji | RRID:SCR_002285 | ||
| Genetic reagent | Danio rerio | Strain AB | RRID: ZIRC_ZL1 |
Additional files
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Supplementary file 1
Details of oligonucleotides used in the study for generation of landing pads and assay constructs, and mapping of site of integration of transgene in landing lines and test lines.
- https://cdn.elifesciences.org/articles/65601/elife-65601-supp1-v1.xlsx
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Supplementary file 2
Overview of transgenic lines generated in the study.
- https://cdn.elifesciences.org/articles/65601/elife-65601-supp2-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/65601/elife-65601-transrepform1-v1.docx
