Generation Of Zebrafish Knock-out Mutant Line Of Ppm1g-AD And A Preliminary Analysis Of Related Biological Functions

Among human diseases,the incidence of neurological diseases has been increasing in recent years.The pathogenesis of most of these diseases is still unclear,and the treatment methods are limited,resulting in more and more patients and families suffering from these.Therefore,for the basic research,the application of constructing experimental models related to human diseases,the means of molecular biology to study the biological functions of certain neural developmental related factors or explore the pathogenesis of related diseases,will have important indicative value for the diagnosis and treatment of clinical neurological diseases.Zebrafish,with the advantages of in vitro fertilization and early embryo transparency,has become an ideal animal model in the field of neurobiology and disease research.With the rapid development and application of gene editing technology,CRISPR/Cas9 technology has emerged at the right moment,and important progress has been made in the application of zebrafish in neurological disease model in the domestic and overseas,which has promoted the development of neurological research field.PP2C protein phosphates are a family of metal-dependent phosphates.Ppm1 g,as one of the members of this family,contains not only the same structural domain as the rest of family members,but also a specific structural domain,which is named the acidic domain(AD),ranging from 138 th aminoacid to 157 th amino acid.Other studies have shown that mice which was complete knocked-out of ppm1 g gene showed a mutant phenotype with abnormal nerve development.However,the function of the conserved AD in Ppm1 g protein in vivo has not ever been reported.The results of bioinformatics analysis indicated that the coding sequence of AD in zebrafish was derived from a 60 bp sequence of the fifth exon of ppm1 g gene located on chromosome 13 of zebrafish.Two target sites were selected in the upstream and downstream of the coding sequence,and subsequently two target gRNAs would be synthesized.Therefore,this research ultimately obtained CRISPR-Ppm1g-AD(-4,+1bp)knockout zebrafish lines,which contains the 4 bp deletion on the upstream target site and 1 bp insertion on the downstream target site of Ppm1g-AD coding sequence,through micro-injecting wide-type zebrafish embryos,detecting validity of the targeted point,testing the heritability of F0 mosaics and obtaining F1 mutant hybrid lines.Further analysis indicated that the successful construction of this zebrafish indel model laid an important foundation for studying whether Ppm1g-AD was related to neural development in zebrafish.Phenotypic analysis of mutant lines showed that different degrees of slow nerve development were observed in Ppm1g-AD(-4,+1 bp)heterozygotes and homozygotes,such as midbrain retardation.Meanwhile,in situ hybridization analysis showed that: compared to the wide-type,the expression of neural developmental gene pax2 was down-regulated and notch1 b was up-regulated inmutant homozygous lines.These above results have showed that the Ppm1g-AD knocked-out zebrafish lines led to abnormal nerve development,which were of great significance for exploring the function and mechanism of Ppm1 g and its AD in the process of nerve development in zebrafish.