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.
Highlights
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
The limitations of the Cre/LoxP technology outlined above, are mainly related to the weak and often variable expression of promoters in transgenes containing the Cre or CreERT2 coding sequences, which frequently do not induce the deletion of all different types of floxed alleles in the cells where they are expressed (Fig. 1a, b)
We sought to develop a new DNA construct that would be easy to induce by Cre/CreERT2-mediated recombination, and that would subsequently enable strong and sustained co-expression of a fluorescent reporter and a constitutively active Cre (Fig. 1c)
Summary
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. Most mouse genes have been flanked by LoxP sites (floxed), and mouse lines are available expressing constitutively active Cre or tamoxifen-inducible CreERT2 in almost any cell type[1,2,3] The availability of these genetic resources enables the precise conditional deletion of almost any mouse gene in any cell type and at a defined time-point, which are crucial requirements for understanding the function of genes during organ development and disease. Scientists have generated reporters of Cre/LoxP recombination, and these have become widespread and essential genetic tools in any laboratory performing genetic studies These reporters are usually alleles targeted to the ubiquitous mouse ROSA26 locus[11,12] and are activated or expressed only after the cell expresses Cre or has induced CreERT2 activity
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