Fig. 1.

Design and pre-validation of iSuRe-Cre plasmids. a, b Cre or CreERT2 expression from transgenes carrying gene-specific promoters (i.e. Tie2 or Cdh5) is variable and relatively weak (endothelial RNA samples, n = 3 animals). In addition, in the case of CreERT2-expressing alleles, tamoxifen needs to be provided to induce activity, of only a fraction of available CreERT2 proteins, and in a limited time window. Due to the variability in Cre expression and induced activity, commonly used Cre activity ROSA26 reporter alleles do not guarantee full recombination of other floxed alleles in the same cell. c The new iSuRe-Cre genetic tool enables stronger (CAG promoter driven) and constitutive co-expression of a reporter (MbTomato) and Cre, which is expected to increase the correlation between reporter expression and gene deletion. LoxP, short DNA sequences recognized and recombined by Cre; N-PhiM, nuclear-localized and mutated PhiYFP; pA, Sv40 polyadenylation sequence; 2 A, self-cleaved viral 2 A peptide to guarantee equimolar expression but separate localization of the reporter and Cre. WPRE, Woodchuck hepatitis virus posttranscriptional regulatory element to enhance expression. d Vectors containing the normal Cre sequence always had deletion of the N-PhiM-pA cassette in E. coli, rendering them non-inducible. e The use of an intron-containing Cre sequence (Int-Cre) disrupts Cre expression and function in E. Coli, allowing the generation of a Cre-inducible, Cre-expressing vector (iSuRe-Cre). f Confocal micrographs showing that cells transfected with iSuRe-Cre plasmids express the N-PhiM protein in the nuclei. If cells are co-transfected with iSuRe-Cre and Cre-expressing plasmids, the LoxP-N-PhiM-pA-LoxP cassette is recombined/deleted and MbTomato-2A-Int-Cre is expressed. Scale Bars 55μm. Error bars indicate StDev. Source data are provided as a Source Data file