| (?)Ferrets have served as one of the most valuable animal models for recapitulating human disease since 1933.Because of they show many similarities to humans in terms of physiological features of brain function and reproductive biology as well as pathological characteristics of various diseases like Cancer,Influenza,and Vesicular Fibrosis.Despite these advantages,the current use of ferrets in research is significantly limited due to the lack of method enabling precise genetic modification.Ferret as an animal model also has its own uniqueness in research.Firstly,there is already a gyrus structure in the cerebral cortex of the ferret,which makes the ferret more close to humans in brain development and structure than mouse;the second is the shorter reproduction cycle and stronger fertility,also the individual's availability is easier than non-human primates.It has advantages in the convenience of experiment operation and experiment cost.We used the CRISPR/CAS9 system by co-injecting CAS9 mRNA and sgRNA into ferret 1-cell embryos,changing the alleles in candidate genes(Doublecortin,DCX)which resulting in mutations in this genes.Highly efficient genetically modified ferrets can be successfully obtained,and their efficiency can be as high as 73.3%.DCX play an important role in brain development and the generation of cerebral cortex sulcus.In human with DCX mutation,their cerebral cortex becomes smooth and the ventricles are enlarged,which is what we call the "smooth brain".In DCX-mutated ferrets,the cerebral cortex also becomes smooth,accompanied by thickening of the cerebral ventricle,increased cortical thickness,and changes in neuronal cell morphology.These phenotypes are very similar to human patients.Further,the number of neuron in DCX-mutated ferrets is increased and neuron migrated to wrong place in cortex,the same to the neural precursor cell.As far as radial glial(RG)cell,the arrangement and the structure of radial glial basal process,a scaffold for neuronal migration,are disorganized in mutant cortices.In addition,we also build a complete set of female jill reproductive cycles and effective ferret induced superovulation programs.Formed a complete ferret surgical operation including ligation and embryo transfer.Our study demonstrates the CRISPR/CAS9 system is a highly specific,efficient and multi-site genetic editing method among eukaryotes.It has also been highly efficient,precise,and widely expressed in ferrets.In combination with the phenotype of mutated ferrets under MRI,we demonstrate for the first time that CRISPR/CAS9-mediated gene editing allows for efficient generation fo gene knockout ferrets.In the future,ferret may be a kind of model animal for human brain development diseases.(?)During evolution,neural progenitor cells in the subventricular zone(SVZ)have fundamental functions,ranging from brain volume expansion to the generation of a 6-layered neocortex.In lissencephalic animal models,such as rodents,the majority of neural progenitors in the SVZ are intermediate progenitor cells(IPCs).Most IPCs in rodents undergo neurogenic division,and only a small portion of them divide a very limited number of times to generate a few neurons.Meanwhile,in gyrencephalic animals,such as primates,IPCs are able to self-renew for up to five successive divisions.However,abundant IPCs with successive proliferative capacity have not been directly observed in non-primate species.In this study,we examined the development of neural progenitors in the Chinese tree shrew(Tupaia belangeri chinensis),a lissencephalic animal with closer affinity than rodents to primates.We identified an expansion of the SVZ and the presence of outer radial glial(oRG)cells in the neocortex.We also found that IPCs have the capacity to self-amplify m ? Ltiple times and therefore serve as major proliferative progenitors.To our knowledge,our study provides the first direct evidence of abundant IPCs with proliferative potential in a non-primate species,further supporting the key role of IPCs in brain expansion. |
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