Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression.

The laboratory rabbit has been a valuable model system for human disease studies. To make the rabbit model more amendable to targeted gene knockin and stable gene over-expression, we identified a rabbit orthologue of the mouse Rosa26 locus through genomic sequence homology analysis. Real-time PCR and 5' RACE and 3' RACE experiments revealed that this locus encodes two transcript variants of a long noncoding RNA (lncRNA) (rbRosaV1 and rbRosaV2). Both variants are expressed ubiquitously and stably in different tissues. We next targeted the rabbit Rosa26 (rbRosa26) locus using CRISPR/Cas9 and produced two lines of knock-in rabbits (rbRosa26-EGFP, and rbRosa26-Cre-reporter). In both lines, all the founders and their offspring appear healthy and reproduce normally. In F1 generation animals, the rbRosa26-EGFP rabbits express EGFP, and the rbRosa26-Cre-reporter rabbits express tdTomato ubiquitously in all the tissues examined. Furthermore, disruption of rbRosa26 locus does not adversely impact the animal health and reproduction. Therefore, our work establishes rbRosa26 as a safe harbor suitable for nuclease mediated gene targeting. The addition of rbRosa26 to the tool box of transgenic research is expected to allow diverse genetic manipulations, including gain-of function, conditional knock out and lineage-tracing studies in rabbits.