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.
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