Establishment Of Knockout Mouse Model System For Bone Development-related Genes Based On CRISPR/Cas9 Technology

Purpose:A suitable animal model is a vital tool in the study of bone development.The main purpose of this article is to establish a system based on CRISPR/Cas9 technology to construct knockout mice related to bone development.Method:In the first part,we constructed Phex knockout mice using the conventional CRISPR/Cas9 technology process.For Phex gene,we designed an sgRNA and ligated its transcription template with a pX330 plasmid to construct an expression vector for amplifying the transcription template.The mixed solution of sgRNA and Cas9 mRNA,which was linearized and transcribed in vitro,was injected into the cytoplasm of the fertilized eggs of mice by microinjection technology,and chimeric mice of F0 generation were obtained.F0 generation and WT mice were mated to obtain F1 generation mice.The mice carrying the desired mutation were screened out by gene identification and sequencing.In the second part,we used the optimized CRISPR/Cas9 technology process to construct Emcn knockout mice.Targeting Emcn,we designed two sgRNAs.Then the DNA template fragments of sgRNA can be rapidly amplified by PCR technology,which can be directly used for in vitro transcription.Finally,the mixture of sgRNA and Cas9 mRNA was injected into the cytoplasm of the fertilized eggs of mice according to the routine procedure,and mice carrying the target mutation and stable inheritance were obtained.Result:Phex knockout mice constructed on the basis of conventional technical procedures were cut by Cas9 directed by sgRNA.Due to the small mutation fragments,offspring mice need to be sequenced to identify genotypes.At the protein level,the expression of PHEX protein in Phex knockout mice was significantly reduced.In appearance,Phex knockout mice are short and have curved hind limbs.Micro-CT results showed that the number and surface area of bone trabeculae in the posterior limb of Phex gene knockout mice were significantly reduced,and the space between bone trabeculae was significantly increased.The results of whole blood biochemical analysis showed that the serum phosphorus of Phex gene knockout mice was significantly lower than that of normal mice,and the serum alkaline phosphatase was significantly higher than that of normal mice,while the serum calcium content of Phex gene knockout mice was not significantly different.The Phex knockout mouse can be used as a model organism for the study of XLH disease,as its various phenotypes are similar to those of XLH(X-linked hypophosphatemia)caused by Phex mutations in humans.The Emcn gene knockout mice constructed based on the optimized technical process were directed by two sgRNA to cut both ends of the Emcn by Cas9,so as to achieve the goal of knocking out the Emcn.The genes that were knocked out in mice were so long that they could be genotyped by PCR.Western Blot and immunohistochemical methods were used to measure the expression of EMCN in mice,and the results showed that compared with normal mice,EMCN protein was almost not expressed in Emcn knockout mice,which also verified the successful construction of Emcn gene knockout mice.The successful construction of Phex knockout mice and Emcn knockout mice demonstrated that we successfully established a CRISPR/Cas9-based system for the construction of bone development-related knockout mice.Moreover,the optimized technical process greatly reduced the time of preparation of reagents,which provided experimental operation guidelines for more efficient construction of mouse models related to bone development,and also laid a foundation for more intuitive research on the relationship between genes and bone development diseases.