小鼠模型,医学研究 z-bo 2020-10-22 335

　　The mouse model has become an irreplaceable model animal for biomedical research such as studying gene functions and pathogenic mechanisms, establishing human-related disease models, and evaluating the safety and effectiveness of drugs developed. Translational medicine research is aimed at actual problems from clinical patients, and through laboratory related basic and pre-clinical research including molecules, cells and model animals, it is finally realized to solve the known and unknown problems faced by the clinic. Therefore, the thinking and strategy of translational medicine research is to combine clinical problems through basic laboratory research and preclinical animal experimental research, and then finally transform the research results into clinical practical applications.

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

　　The use of mouse models to carry out preclinical related research is an important bridge to realize the transformation of basic research into clinical application. To establish such a vital connection bridge, building a humanized mouse model is an indispensable strength for preclinical research. Powerful tool. Past research practice tells us that in order to publish high-quality articles, the ideal research strategy is to find potential new genes related to the occurrence of diseases from clinical practice, and construct corresponding gene-edited mouse models to confirm by research. The causal relationship between potential disease-related genes and human diseases and their pathogenesis-related mechanisms lay the foundation for the establishment of mouse models of human-related diseases, and also create conditions for the evaluation of clinical treatment drugs and methods for human-related diseases.

　　Next, I will share the successful case of our client’s high-scoring article "In Vivo AAV-CRISPR/Cas9-mediated Gene Editing Ameliorates Atherosclerosis in Familial Hypercholesterolemia" published in the internationally renowned journal "Circulation" this year to further explain how to apply gene editing tools. Mouse model, develop research ideas, strategies and technical methods on the relationship between related genes and diseases, and how to use the gene-edited mouse model to evaluate the effectiveness of gene therapy for diseases.

　　1. Establishment of mouse disease model of familial hypercholesterolemia

　　The author of the article first screened the low-density lipoprotein receptor (LDLR) from clinically homozygous patients with familial hypercholesterolemia and blood sugar (manifested as elevated plasma levels of high cholesterol and low-density lipoprotein, as well as blood atherosclerosis and other phenotypes). ) Unknown new mutation (such as E207X), and in vitro cell experiments confirmed that the expression of the mutant LDLR gene is related to its functional changes. Then, how to confirm the causal relationship between the newly discovered point mutation and familial hypercholesterolemia and atherosclerosis? The author understands that relying only on the results of in vitro experiments is difficult to be truly convincing. Therefore, the author plans to replicate the human LDLR-E207X point mutation in mice to study the relationship between the new mutation of the gene and the occurrence of diseases. The author sought help from Saiye Biology to construct the LDLR-E207X point mutation mouse model. By comparing human LDLR and mouse Ldlr gene sequences, we found that the corresponding human LDLR E207X point mutation is located at the E208X position of mouse Ldlr. Using CRISPR/Cas9 gene editing technology, we designed the corresponding gRNA series and donor DNA, and through pronuclear microscopy Injection method, successfully constructed Ldlr-E208X point mutation mouse model. The results of the study showed that because the point mutation introduced the stop code, the mouse Ldlr protein expression function was lost, and the mouse model showed obvious hypercholesterolemia and atherosclerotic phenotypes under the induction of high-fat diet. By constructing the Ldlr-E208X point mutation mouse model, the authors not only confirmed the causal relationship between Ldlr-E208X point mutation and clinical patients with hypercholesterolemia and atherosclerosis, but also established a mouse model of the disease.

　　2.AAV-CRISPR/Cas9 gene therapy

　　Based on this mouse model, the author further explored the possibility and effect of gene modification therapy to treat the disease. Using liver-specific serotype AAV8 vector-mediated CRISPR/Cas9 gene therapy technology, by constructing liver-specific expression vectors for AAV8-Cas9 and AAV8-gRNA plus normal Ldlr donors, subcutaneous injections are used to treat newborn Ldlr -E208X point mutant mice, the results confirmed that after AAV8-CRISPR/Cas9 gene therapy, the normal Ldlr protein expression in the mice recovered to a certain extent, and the hypercholesterolemia and atherosclerotic phenotypes of the mice were also obtained A certain degree of improvement indicates that AAV8-CRISPR/Cas9 in vivo Ldlr repair treatment has a certain effect. The author discovered possible new disease-causing genes from clinical patients, to construct mouse models of point mutations, to confirm the causal relationship between point mutations of the gene and the disease, and to prove that AAV8-CRISPR/Cas9 gene therapy can alleviate the disease phenotype. It was verified and finally put forward the hypothesis of the mechanism and treatment of the disease, that is, due to the LDLR E207X point mutation, the function of the gene is damaged, resulting in an increase in blood LDL content, which causes hypercholesterolemia and atherosclerotic phenotype. The AAV8-CRISPR/Cas9 expression vector is used to modify the liver LDLR gene function, which has the effect of lowering LDL in the blood, thereby playing a therapeutic effect on alleviating hypercholesterolemia and atherosclerosis.

　　This article, from research ideas and strategies to technical methods, provides a very good reference for us in the future to apply gene-edited mouse models, carry out related gene function studies, establish disease models, and evaluate treatment methods and their effects. effect.

　　Why build a humanized mouse model? What are the types of humanized mouse models?

　　In the past, when people mentioned humanized mouse models, they first thought of using immunodeficient mice to transplant human cells or tissues to make the mice contain human immune system. 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, transplantation of human intestinal microorganisms and other methods. Therefore, the so-called humanized mouse model refers to the introduction of human cell tissues, human genes or human intestinal microorganisms into mice through transplantation or gene editing technology, and the construction of mice contains human-related cells or tissues. Mouse models such as genes or gut microbes.

　　Why build a humanized mouse model? As humans and mice are different species, although there are similarities in genetics, anatomy, physiology, pathology, and metabolism, there are naturally many differences, especially the obvious differences in their immune systems, which restricts some people. Research on the pathogenic mechanism of pathogenic pathogens and the interaction of anti-infection/tumor microenvironment, such as HIV virus, dengue virus, hepatitis virus, coronavirus, etc., infect human viruses, but mice are completely or very unsusceptible. By transplanting human-derived cells into immunodeficient mice, a humanized immune system mouse model is constructed, making this type of mouse model a susceptible model animal for human virus infection, which is helpful to establish a small study of human virus pathogen infection Rat model. 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 accurate clinical tumor treatment. However, how to truly reflect and evaluate the anti-tumor effects of human immune cells and tumor microenvironment The role played 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, the construction of a humanized mouse model containing human immune cell system is effective in validating and evaluating anti-tumor immunity. Played a vital role in the 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 allowed people to see the huge development prospects for the development of such human-derived therapeutic antibodies. Because anti-ICP human antibodies are directed against human targets, if you plan to use mouse models to verify and evaluate the safety and effectiveness of anti-ICP human antibodies in preclinical in vivo, gene editing technologies, such as CRISPR/ Cas9 or TurboKnockout (an improved version of ES targeting established by Saiye Biology) technology, humanized modification of mouse related antibody target genes, and established a gene-edited humanized ICP mouse model. At present, we also provide related ICP humanized commercial mice, including hCTLA-4, hPD-1, hGITR, hVISTA, hOX40, hCD28 and hCD39.

　　In addition, the human intestinal microbiome has been considered to be a component of the human super organism and one of the important organs for maintaining the ecological balance of the human body. Recently, the concept of intestine-brain axis and intestine-liver axis has been put forward, and it has been increasingly explained that the difference and diversity of human intestinal microbial composition directly or indirectly affect human health and the development of diseases, as well as the role of drugs in treating diseases. . By transplanting human intestinal microbiome to sterile mice, constructing a mouse model of humanized intestinal microbes will undoubtedly help to study the causal relationship between intestinal microbes and disease, the pathogenic mechanism, and the influence of drug treatment on special microbiota And other related fields.

　　The characteristics and advantages of BRGSF new immunodeficiency mice?

　　By transplanting human cells/tissues into immunodeficient mice, the human immune system is reconstructed in mice. In the traditional sense, it was first called humanized mouse model, and immunodeficient mice are of this kind of human origin. The basis for the construction of chemical mouse models. In the early 1960s, immunodeficient mice started from early T cell deficient nude mice and experienced T cell and B cell deficient SCID mice. T cell, B cell and some innate immune cell deficient NOD-SCID mice Mice, developed on the basis of NOD-SCID genetic background, by knocking out IL-2rg receptors, respectively constructed more severe immunodeficiency (T/B/NK cell defect) NOG (2002) and NSG (2005) ) An important milestone in mice. In addition, on the basis of BALB/c genetic background mice, by knocking out Rag2 and IL-2rg receptors, BRG mice similar to NOG/NSG immunodeficiency were constructed (2005).

　　In recent years, in order to improve the transplantation efficiency of human myeloid cells in mice, people have introduced human-related cytokines that are conducive to the development and maturation of human myeloid cells on the basis of NOG/NSG mice, such as GM-CSF, IL-3 and SCF have constructed modified immunodeficient mice of NOG-EXL and NSG-SGM3 respectively. Although NOD-SCID mice, which are the basis for NOG/NSG construction, take advantage of the partial innate immune deficiency and Sirpa gene mutation characteristics of NOD mice, which are conducive to the advantages of human cell transplantation, NOD-SCID genetic background mice also have obvious advantages. For example, the loss of complement 5 gene function in NOD-background mice makes it impossible to achieve complement-dependent cytotoxic killing (CDC); while SCID-background mice exhibit increased sensitivity to radioactivity, making them resistant to certain genes Toxic drugs are more sensitive and prone to spontaneous tumor formation. In addition, because NOG-EXL and NSG-SGM3 modified immunodeficiency mice introduce overexpressed human-related cytokines, after transplantation of human-derived cells in such immunodeficiency mice, it is easy to cause mouse anemia, thereby shortening the human source The window period for the use of chemical mouse models (generally only 3 to 7 months).

　　BRG immunodeficiency mice are mice based on BALB/c genetic background. Because their Sirpa gene cannot bind to CD47 on the surface of human monocytes/macrophages, they form a "do-not-eat me" phenomenon, which is not conducive to improving humans. Source cell transplantation efficiency. However, because BRG mice directly knock out the Rag2 gene to achieve mouse T/B cell function defects, thereby overcoming the shortcomings caused by the destruction of SCID genes. By successfully introducing NOD-Sirpa into BRG genetic background mice, the BRGS mice were established to overcome the shortcomings of the original BRG mice and greatly increase the efficiency of transplanting human cells. And on the basis of BRGS mice, knocking out the mouse Flt3 gene related to the development and maturation of myeloid cells, the successfully constructed BRGSF new immunodeficiency mouse (2017) shows its unique advantages.

　　Compared with the current similar NOG-EXL and NSG-SGM2 modified immunodeficiency mice, the transplantation efficiency of human-derived cells (such as human CD34+ hematopoietic stem cells), especially human myeloid cells (such as DC cells), and human-derived cells In terms of effective survival time in mice, the performance of BRGSF new immunodeficiency mice has been significantly improved, coupled with the absence of complement 5 and SCID gene deletion in BRGSF mice, ensuring that immunodeficiency mice can be used as an effective strategy for evaluating the role of CDC. Possibility of pre-clinical research. In addition, because transgenic technology was not used to introduce human-related cytokines (such as GM-CSF and IL-3, etc.), it also avoided the possible anemia in transplanted mice, so that the reconstructed human immune system was in BRGSF mice. It can be maintained for at least one year, which greatly improves the use efficiency of 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 myeloid development research. In addition, because of the high efficiency of implanting artificial blood cells into BRGSF, it is the best model for preparing humanized immune system (HIS) mice to study and predict the immune response in humans. At present, our company can provide BRGSF immunodeficiency mouse products and related technical services for scientific research and medical research and other related research fields. We believe that the successful construction of BRGSF new immunodeficient mice will greatly promote the application of humanized mouse models in translational medicine research.