New creation of humanized animals by genome editing technology
Genetically modified animals play an important role in the study of gene functions and for understanding the mechanisms of human diseases. Genome engineering, such as gene knockout or gene knock-in, in zygotes is achieved by the CRISPR/Cas system via the error prone non-homologous end joining (NHEJ) pathway or via homologous recombination (HR), respectively.
We have so far reported the efficient generation of ssODN-mediated knockins (KIs) in animals (Yoshimi et al. Nat Commun, 2014). We have also generated GFP-KI animals at the targeted Rosa26 loci, and replaced the animal endogenous genes with the human extrinsic genes as 'humanized animals' by the CRISPR/Cas system (submitted).
These technologies facilitate easy and flexible gene editing in living organisms, such as the production of rainbow animals with colorful reporter genes or cross-species genetically modified (e.g. humanized) animals.
Genome engineering technologies in rats
1) gene-driven ENU mutagenesis
2) zinc finger nuclease (ZFN）
3) transcription activator-like effector nuclease (TALEN)
4) clustered regularly interspaced short palindromic repeats / CRISPR associated proteins (CRISPR/Cas9)
The Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University supports external research projects to generate knock-out (KO) and knock-in (KI) rats by the genome engineering technologies.
We also link with the National Bio Resource Project-Rat
1) Severe combined immunodeficiency (SCID) rats
We have so far generated severe combined immunodeficiency (SCID) rats with the genome engineering technique.
- F344-Il2rgem7Kyo (Mashimo T, PLoS ONE 2010)
- F344-Prkdc em2Kyo (Mashimo T, Cell Reports 2012)
- F344-Rag2 em1Kyo (unpublished)
2) Reporter rats
- F344-Rosa26 em1(CAG-GFP)Kyo (unpublished)
3) Human disease models/humanized rats