iCre toolkit and cloning strategy schematics. (A). The top of the schematic shows the gateway-compatible 469-pME-iCre clone (Addgene #171792) encoding the codon-improved iCre as described in the text. This clone could be used to either generate mRNA for injection and rapid experiments (Left side, Transient) or to generate transgenes for further genomic integration and generation of iCre-Driver transgenic lines. On the left side of the upper panel (transient), optimised material for a 2-ways R1/R3 LR-rection using 02-p3T3TS_R1R3, easing success rate of the reaction and designed to improve downstream mRNA synthesis and stability. On the right side, optimised material for 2-ways R4/R2 LR reaction and transgene generation. Each pDest presents miniTol2 sequences complexed with I-SceI sequences and either a Multi Cloning Site (MCS) for rapid integration of any marker of choice or a GFP/mCherry sequence under the control of a Crystallin promoter (for Lens expression and transgenics identification). In this study, we generated the iCre-Driver construct/lines #171797, with iCre under the control of the Ubiquitin promoter (GFP Lens expression as selection marker). (B). Schematic representation of the Lox-Responder sensor “UBI:LoxSENSOR” used in this study. In the absence of iCre, BFP is translated/expressed and terminated with a polyA signal. In the presence of iCre, Lox sequences are recombined, the BFP cassette is removed, triggering translation/expression of a mKate cassette. Ubiquitous expression of iCre protein, via exogenous injection or transgenic expression, should turn the animals from blue to red. Partial or mosaic conversion would be observed via the remaining BFP/Blue expression. See Table 1 for details of each plasmid.

Viability of embryos and recombination efficiencies with iCre-mRNA, Cre-protein or transgenics. (A), Titration of iCre-mRNA injections. Yolk injection of 50 ng/µl, 100 ng/µl, 150 ng/µl, and 300 ng/µl iCre-mRNA, 1 nl Control and uninjected controls (UIC). (B), Titration of Cre protein injections. Cell injection of 0.01, 0.1, 0.5, 1, and 10 ng Cre protein, 2 nl control and uninjected control (UIC). (C), F2 Clutch viability with transgenic iCre expression from F1 iCre-Driver tg(UBI:iCre). (D), Quantification of Lox-recombination following iCre-mRNA injections. Lox-recombination has been assessed based on the detectable conversion/shift of BFP fluorescence to mKate fluorescence. Partial Lox-conversion is evidenced by the combined expression of both BFP and mKate fluorescence, while full Lox-conversion is evidenced by mKate expression with no detectable BFP fluorescence. Results are presented as percentages. (E), Quantification of Lox-recombination following Cre-protein injections. Cre-protein injections ranging from 1 ng to 10 ng were sufficient to trigger robust and reproducible conversions. (F), Quantification of Lox-recombination with transgenics. Female iCre-driver systematically triggered complete and homogeneous Lox-recombination, suggesting a robust effect of iCre mRNA and/or iCre protein maternal deposition.

iCre-mRNA injections successfully trigger Lox-recombination but still with some visible partial conversions. (A–C), Control injections demonstrating no spontaneous Lox-recombination in absence of iCre. (D–L), Injection of 50pg–300 pg of iCre mRNA at one-cell stage is enough to promote Lox-recombination. However, partial conversion is still observed with the line studied. At 300 pg, most animal (81%) were however successfully fully converted with no visible blue fluorescence. Scale bar 100 µm.

Female iCre-driver tg(UBI:iCre) triggers 100% recombination of Lox-Responder transgenes. (A–C), When our Lox-Responder tg(UBI:LoxSENSOR) is crossed against a wildtype control, no Lox-recombination is observed and all analysed animals presented with homogeneous BFP/blue fluorescence. (D–F), When Females tg(UBI:iCre) are crossed against Males tg(UBI:LoxSENSOR), 100% all clutches observed presented with homogeneous and fully converted Lox-transgenes; i.e., all animals presented Red/mKate fluorescence without any visible Blue/BFP fluorescence, evidencing complete Flox-recombination in all animals observed. (G–I), When Males tg(UBI:iCre) are crossed against Females tg(UBI:LoxSENSOR), >70% of animals presented with homogeneous full Lox-recombination (Figure 2E), but a few animals remain only partially floxed, evidenced by the presence of around 30% of animals presenting both Red/mKate and Blue/BFP fluorescence. Scale bar 100 µm.

Cre protein injections successfully trigger Lox-recombination. (A–C), Control injections demonstrating no spontaneous Lox-recombination in the absence of Cre protein. (D–R), Injection of 10 pg to 10 ng of cell-permeant Cre protein at one-cell stage. No significant Lox-recombination is observed from 10pg to 100 pg. However, robust conversions are observed from 0.5 ng to 10ng, with 100% of the animal injected with 10 ng presenting total conversion of the Lox-transgenes (i.e., only Red/mKate fluorescence was detected). Note that protein injections may also be an attractive approach for local Lox-recombination at a later stage of zebrafish development. Scale bar 100 µm.