小鼠模型 z-bo 2020-10-16 298

　　The mouse model has become an irreplaceable model animal for biomedical research, such as the study of genetic functions and pathogenic mechanisms, the establishment of human-related disease models, and the evaluation of the safety and effectiveness of developed drugs. Translational medicine research addresses the actual problems of clinical patients, and through laboratory-related basic and preclinical research involving molecules, cells and model animals, it finally solves the known and unknown problems faced in clinical practice. Will come true. Therefore, the thinking and strategy of translational medicine research is to combine basic laboratory research and preclinical animal experiment research to transform the research results into clinical practical applications.

　　How to use the mouse model to send high-quality articles?

　　The use of mouse models for preclinical research is an important bridge for the transition from basic research to clinical application. Establishing a humanized mouse model is essential for establishing such an important bridge for preclinical research. Powerful tool. In past research, if you want to publish high-quality articles, the ideal research strategy is to discover potential new genes related to disease development from clinical practice, and construct corresponding gene-edited mouse models in the research. Yes confirm. The causal relationship between potential disease-related genes and human diseases and their etiological-related mechanisms have laid the foundation for establishing mouse models of human-related diseases and evaluating clinical treatment drugs and methods for human-related diseases. Create conditions.

　　Once again, by sharing the successful case of your high-scoring article "InVivo AAV-CRISPR/Cas9-MediatingGeneEditingAmelioratesAtheroclerosis in FamilyHolepercholesterolemia" published in the internationally renowned magazine "Circulation" this year, you can share related gene-edited mouse models. Learn more about how to apply and run. The relationship between diseases, and methods of using gene-edited mouse models to evaluate the effectiveness of gene therapy on diseases, as well as studying other aspects of thinking strategies and technical methods.

　　1. Establish a mouse disease model of familial hypercholesterolemia

　　The author of the article suffers from familial hypercholesterolemia (manifested by elevated plasma levels of high cholesterol and low-density lipoprotein, and blood atherosclerosis). We first studied clinically homozygous patients such as phenotypic sclerosis, screened new unknown mutations in low-density lipoprotein receptor (LDLR) (E207X, etc.), and performed mutant LDLR through in vitro cell experiments. We confirmed that gene expression is related to changes in its function. So how do we determine the causal relationship between newly discovered point mutations and familial hypercholesterolemia and atherosclerosis? The authors understand that it is difficult to rely solely on the results of in vitro experiments, so they plan to replicate human LDLR-E207X point mutations in mice and study the relationship between new gene mutations and disease development. Yes. The author turned to Saiye Biology to establish a LDLR-E207X point mutation mouse model. By comparing the gene sequences of human LDLR and mouse Ldlr, it is found that the corresponding human LDLRE207X point mutation is located at the E208X position of mouse Ldlr. Using CRISPR/Cas9 gene editing technology, the corresponding gRNA series and donor DNA were designed and injected under the pronuclear microscope. The Ldlr-E208X point mutation mouse model was successfully constructed. The results of the study showed that the point mutation introduced a stop code, resulting in the loss of mouse Ldlr protein expression function, and the mouse model showed obvious hypercholesterolemia and atherosclerotic phenotypes under the induction of a high-fat diet. Display the displayed content. By constructing a mouse model of the Ldlr-E208X point mutation, the authors not only confirmed the causal relationship between the Ldlr-E208X point mutation and clinical hypercholesterolemia and atherosclerosis patients, but also confirmed the disease. A mouse model was also established.

　　2.AAV-CRISPR/Cas9 gene therapy

　　Based on this mouse model, the author will further study the potential and efficacy of gene modification to treat the disease. The CRISPR/Cas9 gene therapy technology was used to construct AAV8-Cas9 and AAV8-gRNA liver-specific expression vectors through liver-specific serotype AAV8 vectors, and normal Ldlr donors were injected subcutaneously to treat the newborn Ldlr. -E208X point mutant mice, the results showed that after AAV8-CRISPR/Cas9 gene therapy, the expression of Ldlr protein in mice returned to normal, and the phenotype of hypercholesterolemia and atherosclerosis was obtained. I confirmed it. Some improvements indicate that AAV8-CRISPR/Cas9 in vivo Ldlr repair therapy has a specific effect. The author discovered genes that may cause new diseases in clinical patients, constructed a point mutation mouse model, confirmed the causal relationship between the gene and point mutations in the disease, and AAV8-CRISPR / We have proven that Cas9 gene therapy can reduce The phenotype of the disease. We tested the mechanism and treatment hypothesis of the disease, and finally proposed it. That is, the LDLRE207X point mutation impairs gene function, increases the LDL content in the blood, and causes the phenotype of hypercholesterolemia and atherosclerosis. The AAV8-CRISPR/Cas9 expression vector is used to modify the function of the LDLR gene in the liver. It has the effect of lowering LDL in the blood, and has a therapeutic effect on reducing hypercholesterolemia and atherosclerosis.

　　From research ideas and strategies to technical methods, this article applies gene-edited mouse models, conducts related genetic function studies, establishes disease models, and future treatment methods and their effects. It provides a good evaluation method. Reference role.

　　Why build a humanized mouse model? What are the types of humanized mouse models? When humanized mouse models were mentioned in the past, the first idea was to use immunodeficient mice to transplant human cells or tissues and incorporate the human immune system into the mice. However, the current concept of humanized mouse models has been extended to include the application of gene editing technology, the introduction of human-related genes or the application of sterile mice, the transplantation of human gut microbes, and other methods. Therefore, the so-called humanized mouse model refers to the introduction of human cell tissues, human genes or human gut microbes into mice through transplantation or gene editing technology, and the mouse construction involves human-related cells or tissues. Mouse models, such as genes and gut microbes.

　　Why build a humanized mouse model? Since humans and mice belong to different species, there are similarities in genetics, anatomy, physiology, pathology, and metabolism, but of course there are many differences. In particular, there are obvious differences in the immune system, which limits some people. Study the pathogenic mechanism of pathogens and the interaction of anti-virus/dengue virus, hepatitis virus, coronavirus and other anti-infective/tumor microenvironment, infect human virus, but mice are completely or very insensitive. By transplanting human-derived cells into immunodeficient mice, a humanized immune system mouse model was constructed. This type of mouse model became a susceptible model animal for human virus infection and established a small human virus pathogen infection. Scale research. Useful for rat models. The PDX mouse model established by transplanting tumor tissues of tumor patients into immunodeficient mice has also become an important strategy and technical method for precise clinical treatment of tumors. However, how to truly reflect and evaluate the anti-tumor effects of human immune cells and tumor microenvironment in immunotherapy requires the establishment of a more effective mouse model of the human immune response system. For example, the anti-tumor effects of CAR-T and immune checkpoint inhibitors are inseparable from the participation of related immune cells. Therefore, we have effectively verified the anti-tumor immunity by constructing a humanized mouse model containing human immune cell system. And can be evaluated. It plays an important role in treatment. In recent years, the success of anti-CTLA-4 and anti-PD-1 antibodies as immune checkpoint (ICP) inhibitors in clinical anti-tumor immunotherapy has broad prospects for the development of such human-derived therapeutic antibodies. To people. Since anti-ICP human antibodies target human targets, mouse models should be used to verify and evaluate the safety and effectiveness of anti-ICP human antibodies in preclinical in vivo using CRISPR/isogenes. Editing technology Cas9 or Turbo Knockout (an improved version of ES targeting established by Saiye Biology) technology, humanized modification of mouse-related antibody target genes, and establishment of a gene-edited humanized ICP mouse model. We currently also provide related ICP humanized commercial mice, such as hCTLA-4, hPD-1, hGITR, hVISTA, hOX40, hCD28, hCD39. In addition, the microbiome of the human intestine is a component of human super life and is considered to be one of the important organs for maintaining the ecological balance of the human body. In recent years, the concepts of gut-brain axis and gut-hepatic axis have been put forward. The difference and diversity of human gut microbial composition directly or indirectly lead to the onset of human health and disease, as well as drugs to treat diseases. People are increasingly explaining that it affects characters. .. The construction of a mouse model of humanized intestinal flora by transplanting human intestinal flora into sterile mice is undoubtedly the causal relationship between intestinal flora and diseases, pathogenic mechanisms and characteristics. Help to study the effects of drug treatments on various microflora and other related fields.

　　What are the characteristics and advantages of BRGSF's new immunodeficiency mice? By transplanting human cells/tissues into immunodeficient mice, the human immune system is rebuilt in the mice. In the traditional sense, it was originally called a humanized mouse model, and immunodeficient mice belong to this type. The basis for building a humanized mouse model. In the early 1960s, immunodeficient mice started from the early T-cell-deficient nude mice and experienced T-cell and B-cell-deficient SCID mice. T cells, B cells and certain innate immune cell deficient NOD-SCID mice are based on the genetic background of NOD-SCID, and mice developed by knocking out IL-2rg receptors are more severe. Established immunodeficiency (T/B/NK cell deficiency) NOG (2002) and NSG (2005). ) Is an important milestone for the mouse. In addition, based on the Rag2 and IL-2rg receptor gene knockout of BALB/c genetic background mice, BRG mice with immunodeficiency similar to NOG/NSG were constructed (2005). In recent years, in order to improve the transplantation efficiency of human bone marrow cells in mice, human-related cytokines that promote the development and maturation of human bone marrow cells have been introduced based on NOG/NSG mice such as GM-. CSF, IL-3, SCF, etc. constructed NOG-EXL and NSG-SGM3 modified immunodeficient mice respectively. OD-SCID mice are the basis for the construction of NOG/NSG. It utilizes some of the characteristics of congenital immunodeficiency and Sirpa gene mutations in NOD mice to promote the benefits of human cell transplantation, but the advantages of NOD-SCID. Mice with genetic background also have obvious benefits. For example, the loss of complement 5 gene function in NOD background mice makes it impossible to achieve complement-dependent cytotoxic killing (CDC), but SCID background mice become more sensitive to radioactivity and certain genes. It is resistant. Toxic drugs are more sensitive and prone to spontaneous tumors. In addition, NOG-EXL and NSG-SGM3 modified immunodeficient mice can introduce over-expressed human-related cytokines, and transplantation of human-derived cells into these immunodeficient mice can cause anemia in the mice. Shorten human resources. The window period for using chemical mouse models (usually only 3-7 months). BRG immunodeficient mice are mice based on BALB/c genetic background. Sirpa gene cannot bind to CD47 on the surface of human single spheres/macrophages, thus forming a "don't eat" phenomenon, which will not lead to improvement. Human-derived cell transplantation efficiency. However, BRG mice directly knocked out the Rag2 gene to achieve the defect of mouse T/B cell function, thereby overcoming the shortcomings caused by the destruction of SCID gene. By successfully introducing NOD-Sirpa into BRG genetic background mice, BRGS mice have been established to overcome the shortcomings of the original BRG mice and significantly improve the efficiency of human cell transplantation. Based on BRGS mice, the successfully constructed BRGSF new immunodeficiency mouse (2017) deleted the mouse Flt3 gene related to the development and maturation of bone marrow cells, showing its unique advantages. Compared with similar current NOG-EXL and NSG-SGM2 modified immunodeficient mice, human-derived cells (such as human CD34 + hematopoietic stem cells), especially human bone marrow cells (such as DC cells) and human-derived transplants effectiveness. BRGSF's new immunodeficient mice have significantly improved performance in terms of effective survival of mouse cells, and because BRGSF mice lack complement 5 and SCID gene deletion, immunodeficient mice are effective in CDC. Can be used for evaluation. The possibility of preclinical research related strategies. In addition, no transgenic technology was used to introduce human-related cytokines (such as GM-CSF and IL-3), which avoided possible anemia in transplanted mice and rebuilt the human immune system of BRGSF mice. Yes. It can be maintained for at least one year and will greatly improve the efficiency of using humanized mouse models.

　　BRGSF mouse model is a valuable tool for antibody drug development, vaccine development, the efficacy and safety of chimeric antigen receptor (CAR) T cell therapy, and bone marrow development research. In addition, the high efficiency of transplanting artificial blood cells into BRGSF makes it the best model for preparing humanized immune system (HIS) mice for research and prediction of human immune response. Today, our Sie Biology can provide this type of BRGSF immunodeficiency mouse products and related technical services for science, medicine and other related research fields. We believe that the successful construction of new BRGSF immunodeficient mice will greatly promote the application of humanized mouse models in translational medicine research.