iSuRe-HadCre is an essential tool for effective conditional genetics

Methods to modify gene function in mice with high spatiotemporal resolution have revolutionized biomedical research, with Cre-loxP technology remaining the most widely used. We previously generated the iSuRe-Cre mouse allele, a dual Cre-reporter line that, by permanently expressing Cre in reporter-expressing cells, provides certainty that these cells recombine other floxed alleles. Here we report the generation, validation, and utility of a second-generation allele, iSuRe-HadCre. In contrast to its predecessor, the new allele is targeted to the ubiquitously expressed Rosa26 locus, shows no leakiness, is significantly brighter and more sensitive to CreERT2 activity, and has no Cre-related toxicity. iSuRe-HadCre achieves this through a novel inbuilt recombinase genetic switch that ensures that cells expressing a fluorescent reporter have transient, but sufficient, expression of Cre. This new tool will be essential for the precise, efficient, and trustworthy analysis of gene function in single cells or entire tissues of mice. Overall design: Liver and heart Endothelial Cells of Control, Cdh5-CreERT2 + iSuReHad-Cre or Cdh5-CreERT2 + iSureCre mice were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of Tomato signal and analyzed using scRNAseq.