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.
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
Dongshan
D
Yang
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
Jun
J
Song
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
Jifeng
J
Zhang
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
Jie
J
Xu
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
Tianqing
T
Zhu
Department of Cardiac Surgery, Cardiovascular Research Center, University of Michigan, Ann Arbor, MI 48109, USA.
Zhong
Z
Wang
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
Liangxue
L
Lai
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.|Department of Cardiac Surgery, Cardiovascular Research Center, University of Michigan, Ann Arbor, MI 48109, USA.
Y Eugene
YE
Chen
D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology; D055879:Gene Knock-In Techniques; D023421:Models, Animal; D059368:RNA Isoforms; D062085:RNA, Long Noncoding / Q000235:genetics*; D011817:Rabbits; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014018:Tissue Distribution
D059368:RNA Isoforms; D062085:RNA, Long Noncoding