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.

Library of insertion constructs.

5’ element (5E), middle element (ME), 3’ element (3E).

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). C-D: Images of a Tg(lyz:mTurquoise2) F0 (C) and F1 (D) larva showing neutrophil response in cyan at 2 days post fertilization.

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). C-D: Images of a Tg(irg1:tdStayGold) F0 (C) and F1 (D) larva showing macrophage-specific tdStayGold expression.

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 h post LPS injection) and a macrophage-specific mCherry signal (e.g. 33 h post LPS injection).

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). E-F: Images of a Tg(ubb:mTurquoise2) F0 (E) and F1 (F) larva showing ubiquitous mTurquoise2 signal.