CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre -loxP-mediated lineage tracing.

The rat is an important laboratory animal for physiological, toxicological and pharmacological studies. Clustered regularly interspaced short palindromic repeats ( CRISPR)/CRISPR-associated 9 (Cas9) is a simple and efficient tool to generate precise genetic modifications in rats, which will promote the accumulation of genetic resources and enable more precise studies of gene function. To monitor Cre- loxP-mediated excision in vivo, we generated a Cre reporter rat strain ( Rosa26-imCherry) by knockin of a Cre reporter cassette at the Rosa26 locus using CRISPR/Cas9. Rosa26-imCherry rats exhibited inducible expression of the mCherry cassette ( imCherry) using the Cre -loxP system, whereas normal rats exhibited ubiquitous expression of eGFP but not mCherry in the whole body. Injection of adeno-associated virus serotype 9- Cre into the hippocampus and skeletal muscle resulted in mCherry expression in virus-infected cells. Cre -loxP-mediated mCherry expression was then evaluated by crossing Rosa26-imCherry rats with transgenic rats ubiquitously expressing CAG- Cre, heart-specific α- MHC-Cre transgenic rats and liver-specific Alb-Cre knockin rats. Finally, using the established system the expression pattern of Cre driven by two endogenous gene promoters ( Wfs1-Cre knockin rat, FabP2-Cre knockin rat) was traced. In summary, we demonstrated excision of the loxP-flanked allele in Rosa26-imCherry rats via activation of mCherry expression in the presence of Cre recombinase. This newly established Rosa26-imCherry rat strain represents a useful tool to facilitate Cre-expression pattern determination and tracing experiments. [ABSTRACT FROM AUTHOR]