ImPaqT, a Golden Gate-based immunological toolkit for zebrafish transgenesis
- Humboldt-Universität zu Berlin, Faculty of Life Sciences, Germany
- Max Planck Institute for Infection Biology, Germany
Figures
Figure 1 with 3 supplements
Basic principles of Golden Gate cloning for Tol2 transgenesis.
Schematic of a Golden Gate cloning reaction using one insert. The insertion construct and backbone vector are assembled in a single-tube reaction using PaqCI and ligase. PaqCI is a type IIS restriction enzyme. Its recognition site and cleavage are indicated. The font color corresponds to the color of the respective feature indicated in the cartoon. The backbone vector consists of Tol2 sites, PaqCI recognition sites (PaqCI) along with 4-base spacer sequences (N4) and two overhangs (O). The insertion construct includes the gene of interest (GOI) flanked by PaqCI, N4, and the same two overhangs. Assembly involves digestion of backbone and insertion constructs by PaqCI producing matching overhangs and ligation through the DNA ligase forming the final assembly product.
Figure 1—figure supplement 1
Overhangs and construction of insertion constructs.
(A) Schematic of primer design for generating a 5E insertion containing overhang sequences O1 (TTCC, orange) and O2 (TTTG, green), 4-base spacer sequence (N4, CCTT/AAGG, blue), and PaqCI recognition site (CACCTGC, pink) with example primer sequence for the 5E ubiquitin B promoter insertion. (B) Schematic of the amplification of genes for generation of 5E, ME, and 3E with primers containing the PaqCI recognition site, N4, and a specific overhang sequence. (C) Schematic of digested insertion elements with specific overhangs.
Figure 1—figure supplement 2
Sequence of the backbone vector.
(A) Schematic of the backbone vector used for all Golden Gate assemblies conducted in this study. The backbone vector consists of an ampicillin resistance (AmpR) and E. coli origin (CoIE), Tol2 sites, PaqCI recognition sites (PaqCI) along with 4-base spacer sequences (N4) and two overhangs (O1 and O4). (B) Complete DNA sequence of the backbone vector. Different parts of the vector are indicated by color corresponding to the schematic in A.
Figure 1—figure supplement 3
Domestication of endogenous PaqCI sites.
(A) Schematic of the 5E ubb promoter construct as an example of an insertion element containing an endogenous PaqCI site. The construct contains PaqCI sites (pink), spacer sequence (N4, blue), and overhang sequences O1 (orange) and O2 (green). Primers were designed to introduce a single base mutation (highlighted in bold and underlined) in the endogenous PaqCI recognition site (red). (B) PCR amplified ubb promoter with mutated endogenous PaqCI site and homology arms for In-Fusion cloning. (C) In-Fusion reaction of the PCR product led to the final 5E ubb promoter construct with the mutated endogenous PaqCI site.
Figure 2 with 1 supplement
Generation of Tg(lyz:mTurquoise2) zebrafish.
(A) Schematic of the Golden Gate assembly of Tg(lyz:mTurquoise2). The backbone vector contains overhang sequences O1 (TTCC) and O4 (AGGA). lyz is a neutrophil-specific promoter containing overhang sequences O1 and O2 (CAAA). mTurquoise2 encodes a bright cyan fluorescent protein with overhangs O2 and O3 (GCTA). ubb:polyA is assembled at the final position within the backbone with O3 and O4. (B) Schematic of the final assembly product of Tg(lyz:mTurquoise2). Images of a Tg(lyz:mTurquoise2) F0 (C) and F1 (D) larva showing neutrophil-specific expression of mTurquoise2 fluorescent protein in cyan at 2 days post fertilization.
Figure 2—figure supplement 1
Verification of the Tg(lyz:mTurquoise2) zebrafish line.
Tg(lyz:EGFP) and Tg(lyz:mTurquoise2) zebrafish were crossed. Images of a double-positive larva are shown in A and images of a Tg(lyz:mTurquoise2) single-positive larva in B. GFP is presented in magenta, CFP signal in cyan, and merge in white.
Figure 3
Generation of Tg(irg1:tdStayGold) zebrafish.
(A) Schematic of the Golden Gate assembly of Tg(irg1:tdStayGold). The backbone vector contains overhang sequences O1 (TTCC) and O4 (AGGA). irg1 is a macrophage-specific promoter containing overhang sequences O1 and O2 (CAAA). tdStayGold encodes a bright green fluorescent protein with overhangs O2 and O3 (GCTA). ubb:polyA is assembled at the final position within the backbone with O3 and O4. (B) Illustration of the final assembly product of Tg(irg1:tdStayGold). Images of a Tg(irg1:tdStayGold) F0 (C) and F1 (D) larva showing macrophage-specific tdStayGold expression.
Figure 4 with 3 supplements
Generation of Tg(irg1:icre-p2A mTurquoise2) zebrafish for lineage tracing of individual macrophages.
(A) Schematic of the Golden Gate assembly of Tg(irg1:icre-p2A mTurquoise2). The backbone vector contains overhang sequences O1 (TTCC) and O4 (AGGA). The macrophage promoter irg1 contains overhang sequences O1 and O2 (CAAA). icre contains overhangs O2 and O3 (GCTA) for the middle position. p2A mTurquoise2 is assembled at the final position within the backbone with O3 and O4. (B) Illustration of the final assembly product of Tg(irg1:icre-p2A mTurquoise2). (C) Image of a Tg(irg1:icre-p2A mTurquoise2) F0 larva showing macrophage-specific mTurquoise2 signal after stimulation with LPS. (D) Images of a Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) larva that was introduced to the Tg(irg1:icre-p2A mTurquoise2) construct at single-cell stage and was treated with PTU and LPS. The images show a macrophage-specific mTurquoise2 signal (e.g. 5 hr post LPS injection) and a macrophage-specific mCherry signal (e.g. 33 hr post LPS injection).
Figure 4—figure supplement 1
A floxed transgene does not reorganize in the absence of Cre.
As a control for testing of Tg(irg1:icre-p2A mTurquoise2) in Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) animals in Figure 4 and Figure 4—video 1 and Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) control larvae were injected with LPS 2 dpf and screened. Images are shown in CFP, mCherry, and merge 5 hr (left) and 33 hr(right) post LPS injection.
Figure 4—video 1
Time-lapse imaging of Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) injected with Tg(irg1:icre-p2A mTurquoise2) zebrafish for lineage tracing of individual macrophages.
The Tg(irg1:icre-p2A mTurquoise2) construct was introduced into fertilized eggs from Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) zebrafish at their single-cell stage. Larvae were treated with PTU and LPS, and time-lapse imaging was performed for 48 hr. The movie shows a macrophage-specific CFP and mCherry signal. Scale bar is 100 µm.
Figure 4—video 2
Time-lapse imaging of Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) control zebrafish.
As a control to the Cre-induced rearrangement of Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) by Tg(irg1:icre-p2A mTurquoise2) in Figure 4 and Figure 4—video 1 and Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry) control larvae were treated with PTU and LPS, and time-lapse imaging was performed for 48 hr with no macrophage-specific CFP and mCherry signal observed in the absence of the iCre construct. Scale bar is 100 µm.
Figure 5
ImPaqT allows for expandability of individual positions while retaining compatibility with already established transposon components.
(A) Schematic of the 5E ubiquitin B (ubb) promoter construct containing PaqCI recognition sites (PaqCI) along with 4-base spacer sequences (N4) and two overhangs (O1: TTCC, O2: CAAA) for positioning at the 5′ end of the assembly construct. Primers were designed to break up ubb into three fragments with new overhang sequences. (B) Schematic of PCR amplified ubb fragments ubb1–3 with PaqCI, N4 and O1 and O1A (TAGC), O1A and O1B (TTGA), and O1B and O2, respectively. (C) Schematic of the Golden Gate assembly of 5E ubb1–3, ME mTurquoise2, and 3E ubb:polyA into the backbone vector. (D) Illustration of the final assembly product of Tg(ubb1-3:mTurquoise2). Images of a Tg(ubb:mTurquoise2) F0 (E) and F1 (F) larva showing ubiquitous mTurquoise2 signal.
Figure 6
ImPaqT allows for rapid transgene generation by Golden Gate assembly from PCR.
(A) Schematic of PCR amplified fragments mpeg1.1, tdTomato, and p2A containing PaqCI, N4 and overhangs O1 (TTCC), O2 (CAAA), O3 (GCTA), and O3B (CTCG) and dsDNA fragment encoding rac2WT or rac2D57N containing O3B and O4 (AGGA). (B) Schematic of the Golden Gate assembly of mpeg1.1, tdTomato, p2A, and rac2WT or rac2D57N into the backbone vector. (C) Illustration of the final assembly products Tg(mpeg1.1.tdTomato-p2A-rac2WT/D57N). Images of a Tg(mpeg1.1.tdTomato-p2A-rac2WT) F0 (D) and Tg(mpeg1.1.tdTomato-p2A-rac2D57N) F0 (E) larva 2 hr post tail wounding showing macrophages.
Tables
Table 1
Library of insertion constructs.
5′ element (5E), middle element (ME), 3′ element (3E).
| Insertion elements | Position | Function | Reference |
|---|---|---|---|
| irg1 | 5E | Infection-inducible macrophage-specific promoter | Hall et al., 2013 |
| mfap4 | 5E | Macrophage-specific promoter | Walton et al., 2015 |
| mpeg1.1 | 5E | Macrophage-specific promoter | Ellett et al., 2011 |
| lyz | 5E | Neutrophil-specific promoter | Hall et al., 2007 |
| ubiquitin B (ubb) | 5E | Promoter for ubiquitous expression | Mosimann et al., 2011 |
| ubb:loxP GFP loxP | 5E | Promoter for ubiquitous expression of floxed GFP for use in combination with Cre | Mosimann et al., 2011 |
| hsp70 | 5E | heat shock protein 70-like promoter for inducible gene expression | Shoji et al., 1998; Halloran et al., 2000; Kwan et al., 2007 |
| lck | 5E | T cell-specific promoter | Langenau et al., 2004 |
| runx1 +23 | 5E | Promoter for expression in hematopoietic stem cells | Tamplin et al., 2015 |
| icre | ME | Cre recombinase for inducible recombination of DNA between loxP sites | Shimshek et al., 2002 |
| (p2A) dLanYFP | 3E | Yellow fluorescent protein | Shaner et al., 2013 |
| mNeonGreen | ME, 3E | Green/yellow fluorescent protein | Shaner et al., 2013 |
| LNGFR | ME | Low affinity nerve growth factor receptor for rapid cell enrichment | Wattrus and Zon, 2020 |
| nitroreductase | ME | Inducible ablation of specific cell lineages | Curado et al., 2007; Curado et al., 2008 |
| kid | ME | Ablation of specific cell lineages | Labbaf et al., 2022; de la Cueva-Méndez et al., 2003 |
| (p2A) tdTomato | ME, 3E | Red fluorescent protein | Shaner et al., 2004 |
| tdTomato CAAX | ME | Red fluorescent protein with membrane localization protein | Walton et al., 2015 |
| (p2A) mTurquoise2 | ME, 3E | Cyan fluorescent protein | Goedhart et al., 2012 |
| mCitrine | ME | Yellow fluorescent protein | Zacharias et al., 2002 |
| tdStayGold | ME | Green fluorescent protein | Hirano et al., 2022 |
| ubb:polyA | 3E | Ubiquitous expression of a SV40 poly A | Walton et al., 2015 |
| polyA-gRNA (Control):U6 | 3E | gRNA targeting control site under control of U6 promoter | Zhou et al., 2018 |
| polyA-gRNA (GFP):U6 | 3E | gRNA targeting GFP under control of U6 promoter | Zhou et al., 2018 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Danio rerio) | *AB wildtype | Zebrafish International Resource Center/laboratory stock | RRID:ZIRC_ZL1, ZFIN ID: ZDB-GENO-960809-7 | Background/wildtype strain |
| Genetic reagent (Danio rerio) | Tg(lyz:mTurquoise2)ber2 | This paper | N/A | New transgenic zebrafish line |
| Genetic reagent (Danio rerio) | Tg(irg1:tdStayGold)ber3 | This paper | N/A | New transgenic zebrafish line |
| Genetic reagent (Danio rerio) | Tg(ubb:mTurquoise2)ber4 | This paper | N/A | New transgenic zebrafish line |
| Genetic reagent (Danio rerio) | Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry)cz1701 | Mosimann et al., 2011 | RRID:ZFIN_ZDB-ALT-110124-1 | Obtained from EZRC |
| Genetic reagent (Danio rerio) | Tg(lyz:EGFP)nz117 | Hall et al., 2007 | RRID:ZFIN_ZDB-ALT-071109-2 | Previously described zebrafish line |
| Chemical compound, drug | LPS | Sigma-Aldrich | Cat. # F3665, CAS No. 93572-42-0 | Stimulation of irg1 promoter |
| Chemical compound, drug | PTU | Sigma-Aldrich | Cat. # P7629, CAS No. 103-85-5 | Reduction of pigmentation |
| Software, algorithm | Fiji/ImageJ | NIH | RRID:SCR_002285 | Image analysis |
Additional files
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Supplementary file 1
Sequences of insertion constructs purchased as dsDNA fragments.
(A) Sequence of runx1 +23 including PaqCI sites, O1 and O2. (B) Sequence of tdStayGold including PaqCI sites, O2 and O3. (C) Sequence of LNGFR including PaqCI sites, O2 and O3. (D) Sequence of rac2WT including PaqCI sites, overhang sequence CTCG (O3B) and O4. (E) Sequence of rac2D57N including PaqCI sites, O3B and O4. (F) Sequence of 3E polyA-gRNA (Control):U6 including PaqCI sites, overhang sequence CGTA (O3C) and O4. (G) Sequence of 3E polyA-gRNA (GFP):U6 including PaqCI sites, O3C and O4.
- https://cdn.elifesciences.org/articles/104182/elife-104182-supp1-v1.docx
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Supplementary file 2
Primer sequences.
PaqCI sites are shown in magenta, 4-base spacer sequences are shown in blue, overhang 1 is shown in orange, O1A in purple, O1B in brown, O2 in green, O3 in pink, O3B in light green, and O4 in turquoise. Bases for mutations are highlighted in red.
- https://cdn.elifesciences.org/articles/104182/elife-104182-supp2-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/104182/elife-104182-mdarchecklist1-v1.docx
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ImPaqT, a Golden Gate-based immunological toolkit for zebrafish transgenesis
eLife 14:RP104182.
https://doi.org/10.7554/eLife.104182.3
