Establishment Of Mouse Model Preparation System Using CRISPR/Cas9 And The Study On The Characteristics Of Pathogenic Infection In Humanized Immunodeficient Mice

BackgroundAs a new gene-editing technology,CRISPR/Cas9[clustered regularly interspaced short palindromic repeats?CRISPR?/CRISPR-associated protein 9?Cas9?]has played an important role in animal model construction,basic medical research and biotechnology innovation.Nowadays,the application of CRISPR has been relatively mature and researchers are increasingly in need of gene-editing mice models.Therefore,the construction of gene-editing mouse model using CRISPR will become a fundamental work of great significance.However,the construction of mammalian gene-editing animal model is expensive and technical,and its construction process is somewhat unfamiliar to researchers.As a result,the establishment and optimization of the preparation system of gene-editing mice model is of certain research significance.NSG(NOD.Cg-Prkdc^scidIl2rg^tm1Wj1)is a kind of Prkdc?Protein Kinase,DNA-Activated,Catalytic Polypeptide?and Il2rg?Interleukin 2 Receptor Subunit Gamma?double-gene knock-out mouse model,which is internationally recognized as the model with the highest degree of immunodeficiency and the most suitability for human cell or tissue transplantation.However,in the transplantation of human stem cells to NSG mice,the percentage of mature human immune cells in the mice was no higher than 40 percent,and even about 20 percent in many cases.Therefore,researchers tend to create new immunodeficiency phenotypes based on humanized mouse model and apply the model to stem cell transplantations and specific pathogen infections.In2015,a transgenic mouse model called HLA-A11/DR1 was successfully constructed.Compared to other humanized mice models,the model has a crowd advantage of China's major tissue compatibility complex?MHC?molecules.Obviously,the creation of immunodeficiency phenotype based on HLA-A11/DR1 will surely play an important role in stem cell transplantation,tumor evaluation and specific pathogen infection research in Chinese population.Objectives1.To establish the preparation system of gene-knockout mouse model using CRISPR/Cas9.2.To construct an immunodeficient mouse model based on the humanized mouse model HLA-A11/DR1 and preliminarily evaluate its immunological characteristics.3.To infect the immunodeficient mouse model with influenza virus and dynamically analyze its immunological characteristics.To explore the application of human immunodeficiency mice in the preparation of infection model.Results1.The preparation system of gene-knockout mouse model using CRISPR/Cas9was successfully established.In this model,large fragment deletion occurs in Rnf126and the deletion can be stably inherited.2.HLA-A11/DR1/Prkdc^-/-mouse model was successfully established,which showed the combined deficiency of specific immune cells,the level of non-specific immune cells of the model increased compensatory.3.A state of pro-inflammatory response was found in HLA-A11/DR1/Prkdc^-/-model through the studies on influenza virus infection.The state is more conducive to the early elimination of the virus compared with the control group.However,it also causes serious damage to lung tissue.In addition,the damage of lung tissue is responsible for the death.ConclusionsIn this study,the preparation system of gene-knockout mouse model based on CRISPR/Cas9 technology was successfully established,the HLA-A11/DR1/Prkdc^-/-humanized immunodeficiency mouse model was successfully constructed with this system and the immune and pathogen infection characteristics of this model were comprehensively analyzed.The results provide new techniques and powerful tools for basic and preclinical research.