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

Abstract

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.

Figure 1. Identification of rabbit Rosa26 locus.

(A) Alignment of the mouse Rosa26 sequence and rabbit locus 103345511 (GenBank: XR_515377.1), the putative rbRosa26 locus. Identity score 85%. (B) Illustration of genomic context of the rabbit locus 103345511 and its flanking regions (screen shot from NCBI website). (C) Schematic diagram of the rbRosa26 gene and 2 transcript variants. Top: rbRosa26 genomic sequence, comprising 3 exons (black boxes), and two introns. Bottom two rows: Two transcript variants rbRosa26V1 (628 bp, XR515376.1) and rbRosa26V2, (377 bp, XR515377.1). (D) Relative RNA expression (mean + SEM) of rbRosa26 transcripts in different adult rabbit tissues by real-time PCR. Data are normalized based on the average expression level of XR515377.1 in the lungs (Cq = 25.87). WAT: white adipose tissue.

Figure 2. Production of rbRosa26-Cre-reporter rabbits.

(A). Illustration of CRISPR/Cas9 mediated knock-in strategy to produce rbRosa26-Cre-reporter rabbits. (B) Summary of knock-in efficiencies. (C) Representative genotyping gel of rbRosa26-Cre-reporter founder (i.e. F0) rabbits. #s: # of corresponding F0 kit. KI: knock-in. M: NEB 1 kb DNA Ladder marker.

Figure 3. Transgene expression profiles at rbRosa26 locus.

Left: EGFP expression in different organs/tissues in rbRosa26-EGFP rabbits. Right: tdTomato expression in different organs/tissues in rbRosa26-Cre-reporter rabbits. F1 generation rabbits were used for analysis in both lines.

Figure 4. Germline transmission of rbRosa26 knock-in rabbit lines.

(A) Summary of germline transmission rates of rbRosa26-EGFP and rbRosa26-Cre-reporter knock in founders. (B) Upper: Representative photos of F1 generation knock-in rabbits; lower: geneotyping gel of the F1 generation knock-in rabbits. #s: # of corresponding F1 kit. KI: knock-in. M. NEB 1 kb DNA Ladder marker.

Figure 5. Cre mediated recombination in rbRosa26-Cre-reporter cells.

(A) Without Cre, rbRosa26-Cre-reporter cells express tdTomato (red). (B) RbRosa26-Cre-reporter cells switch to express EGFP (green) as a result of Cre mediated recombination after successful transfection of pBS185CMV-Cre plasmid. (C) Some residual rbRosa26-Cre-reporter cells still expressing tdTomato after Cre transfection. (D) Merged image of (B,C).