We found that the generation of gene knockout (KO) MSM/Ms mice by the CRISPR/Cas9 system was highly practical. domesticus) and has been established by Dr Kazuo Moriwaki as a strain with a defined genetic background produced by inbreeding 12. This strain belongs to a subspecies ( Mus musculus molossinus) of the laboratory mouse ( M. Here, we employed the CRISPR/Cas9 system to target the nonagouti (a) gene that encodes the agouti protein using a Japanese wild-derived mouse strain, MSM/Ms. However, there have been no reports on genome-editing approaches in wild-derived mice to date. One of their most important advantages is that gene-targeted mice can be generated without the intervening steps of generating ESCs and germ line chimaeras. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 have already been applied successfully to gene targeting in laboratory mice 10, 11. Recently developed genome-editing technologies are contributing significantly to biomedical research by generating animals or cell lines carrying expected mutations. Many wild-derived strains can now be cryopreserved and bred safely using these designated ARTs at our centre and other mouse repository facilities 9. We have recently devised basic ART methods including superovulation, in vitro fertilization (IVF), embryo transfer, and sperm/embryo cryopreservation for wild-derived strains in different subspecies of Mus musculus 8. This is also compounded by the unavailability of stable ESC lines, which have been the main tools for generating targeted genetic modifications. However, targeted genetic modifications in wild-derived strains have been more difficult to achieve in comparison to laboratory strains because they are generally resistant to assisted reproductive technologies (ARTs). Furthermore, a large pool of polymorphisms that discriminate conventional laboratory mice from wild-derived mice might enable more accurate and more efficient quantitative trait locus (QTL) analyses 6, 7.
Thus, we expect that they might provide invaluable information on corresponding diseases in humans 3, 4, 5. Wild-derived strains are relatively introduced genetic resources and are expected to increase the chance of finding novel modifier genes that are responsible for some disease-resistance or disease-prone phenotypes. Therefore, it is desirable to use genetically diverse strains of mice including wild-derived mice for pursuing further genetic and physiological studies. Furthermore, such domesticated mice are more prone to many types of tumours and metabolic diseases, such as diabetes, when compared with other species 1. However, the genetic diversity of classical laboratory mouse strains is thought to be limited because of closed breeding using genetically related ancestral strains 2. Since the 1980s, gene-targeting strategies using embryonic stem cells (ESCs) have made this species the standard experimental model for understanding gene functions and associated disease mechanisms 1. The mouse ( Mus musculus) is the most frequently used mammalian species for biomedical research because of its defined genetic background and the relative ease of carrying out genetic modification. Our findings support the empirical hypothesis that nonagouti is a domestication-linked gene, the loss of which might repress aggressive behaviour. These phenotypes were consistent over subsequent generations.
While homozygous knockout offspring were physiologically indistinguishable from wild-type litter-mates, they showed specific domesticated behaviours: hypoactivity in the dark phase and a decline in the avoidance of a human hand. We obtained three homozygous knockout mice as founders, all showing black coat colour. We targeted the nonagouti (a) gene encoding the agouti protein that is localized in hair and the brain. Here, we report that CRISPR/Cas9-mediated gene targeting can be applied to the Japanese wild-derived MSM/Ms strain ( Mus musculus molossinus).
However, gene targeting using wild-derived mice has been unsuccessful because of the unavailability of stable embryonic stem cells. Wild-derived mice have contributed to experimental mouse genetics by virtue of their genetic diversity, which may help increase the chance of identifying novel modifier genes responsible for specific phenotypes and diseases.
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