iSuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications

Abstract

Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis.

Genetic Tools to Study Cardiovascular Biology

“…However, since there is no genetic linkage between the reporter allele and any other floxed alleles in the cell, the correlation between the recombination of a reporter allele and another floxed allele is often very low. Indeed a recent study showed that some commonly used Rosa26 reporter alleles overreport genetic deletions, whereas others underreport (Fernández-Chacón et al, 2019). In our hands, the commonly used R26-LSL-YFP allele (Srinivas et al, 2001), and particularly the R26-LSL-tdTomato reporter allele (Madisen et al, 2010), are extremely sensitive to Cre/CreERT2 activity, unlike the majority of other tested floxed genes (Fernández-Chacón et al, 2019).…”

Endothelial-to-hematopoietic transition is induced by Notch glycosylation and upregulation of Mycn

“…7c, 7d), we first hypothesized that the survival of Mycn iEC-KO adults and their lack of hematopoietic defects was due to incomplete expression of Tie2-Cre (as shown in Fig1e, 1f) and incomplete Mycn deletion in aortic ECs during EHT. To exclude this possibility that blood in Mycn iEC-KO adults derives from wildtype DA EHT events, we crossed Mycn iEC-KO mice with iSuRe-Cre mice, in which cells expressing MbTomato-2A-Int-Cre have full deletion of any floxed allele 58 . Surprisingly, adult Mycn iEC-KO iSuRe-Cre and control Tie2-Cre iSuRe-Cre mice had similar numbers of Tomato-2A-Cre+ hematopoietic cells (Fig.…”

Generation of a Matrix Gla (Mgp) floxed mouse, followed by conditional knockout, uncovers a new Mgp function in the eye

“…While the mouse’s physiology does not fully mimic that of human, the generation of whole-body gene KOs and of tissue conditional knockouts (cKOs) in this species has led to countless discoveries 3 , 4 . The traditional, more cumbersome methods of generating mouse models by gene targeting embryonic stem cells 5 , 6 have been mostly replaced by highly precise gene editing technology that inserts loxP sites flanking an essential DNA fragment of the target gene and make it available to a Cre-recombinase for its targeted mutation 7 , 8 . This gene editing system, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) further allows the precise insertion of exogenous DNA sequences.…”