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1 application characteristics and prospects of transgenic technology and animal models of transgenic diseases in medical research

1. 1 Advantages and disadvantages of main genetic modification technologies applied to making animal models.

1. 1. 1 transgenic technology This technology directly injects the DNA containing the gene expression framework constructed in vitro into the male pronucleus of the fertilized egg, so that it can be randomly inserted into the genome during the amplification of genomic DNA. Among them, BAC transgene has regulatory elements that can completely preserve gene expression, the upstream and downstream sequences of the gene greatly reduce the influence of sequences around the insertion site, and the number of copies inserted into the genome is low and stable, which are not available in traditional transgene. This technology can use strong drivers to overexpress genes, and can be used to simulate and replicate some diseases caused by gene amplification. For example, many tumors are caused by the amplification of oncogenes. Transgenic technology can also be used to express microRNA targeting one or some mRNA in vivo, and the expression of these genes can be turned off or knocked out, so as to reproduce the diseases caused by the lack of expression of these genes. The main disadvantages of this technology are: first, the randomness of insertion may lead to the expression of exogenous transgene not completely following the original endogenous gene expression model, and may also interfere with the expression of endogenous genes; Second, the efficiency is low.

1. 1.2 Gene transformation technology based on homologous recombination. This technology replaces the original sequence with the modified sequence through homologous recombination, thus achieving the purpose of gene modification. This technology needs to obtain chimeric mice through es cell culture, ES cell recombination screening, blastocyst injection, uterine transplantation and other stages, and then obtain the first transgenic stable mouse from the offspring of chimeric mice. This technology can be used for site-directed mutation of genes, replacement of all or part of sequences leading to loss of gene function, and traditional knockout. Conventional knockout of specific genes can be achieved by combining Cre-loxp and Flp-Frt systems, including tissue characteristics and developmental stage-specific knockout. This technology can simulate and reproduce human or animal diseases caused by gene point mutation, and can turn off the expression of one or part of genes in specific tissues and cells and specific development stages, so as to simulate and reproduce the occurrence and development process of specific diseases related to these tissues and cells or development stages. The main disadvantages of this technology are: limited by the culture technology of es cells (there are only several mouse and rat ES cell lines at present), low acquisition rate of newly established mice, long cycle and so on.

1. 1.3 Genetic modification technology based on artificial endonucleases In recent years, many scholars have found several endonucleases that can specifically recognize certain base sequences from microorganisms and improved them, and developed them into artificial endonucleases. Using this technology, some specific DNA sequences can be accurately identified, combined and specifically excised, so as to promote the insertion of foreign DNA sequences into gaps, achieve the purpose of excising or replacing the original sequences, and realize genetic modification. In recent years, artificial endonuclease technology has developed rapidly, including ZFN, TALEN, CRISPR/Cas9 technology, which has the advantages of simple operation, high modification accuracy, high efficiency, short cycle, no restriction by es cells, and transformation of many intracellular genes. Among them, CRISPR/Cas9 technology has been well developed and applied in model animal Institute of Nanjing University. By using this technology, Xingxu Huang and others successfully achieved gene transformation in various species and germ line cell lines, including human es cells, and successfully achieved gene transformation in mice, rats and monkeys, including conditional knockout transformation. The development and utilization of new technologies, such as CRISPR/Cas9, have improved the success rate of transgene and made it possible to construct animal models of transgenic diseases of various genera and strains.

1.2 Application and characteristics of transgenic animal model in medical research

The application characteristics and prospect of 1.2. 1 in western medicine research Based on the in-depth study of mouse genetic information and the genetic research results of human and mouse diseases, a variety of mouse models that can replicate human and mouse diseases have been constructed by genetic transformation technology, and have been widely used to study and reveal the signal pathways, genes and their regulation involved in the occurrence and development of diseases. Because the occurrence and development of tumor is essentially the process of gene mutation and accumulation of somatic cells or germ cells, the tumor disease model constructed by gene modification technology can reproduce the occurrence and development of various tumors, so it has great application value in tumor medical research and anti-tumor drug development, and has achieved great results. At present, it is known that the process of tumor occurrence and development is a process in which signal pathways such as Ras pathway, WNT pathway or PI3K/AKT pathway are activated, while signal pathways that inhibit cell proliferation such as p53 pathway or Rb pathway are destroyed. On the basis of in-depth and full understanding of these molecular mechanisms, a variety of anti-tumor drugs have been developed. For example, EGFR targeted drugs have been used in clinical anti-tumor therapy. The understanding of the regulation mode of p53 pathway and the related molecular structure contributed to the appearance of MDM2 inhibitor RG7 1 12, which has a good therapeutic effect on some tumors to some extent. In recent years, based on the deep understanding of the molecular mechanism involved in the process of tumor metastasis in mice, it provides a theoretical basis for the development of drugs that can widely inhibit tumor metastasis. At present, most animal models of transgenic diseases come from mice due to technical limitations such as difficulty in culturing embryonic stem cells. The successful application of CRISPR/Cas9 technology in a variety of model animal makes it possible to carry out more extensive animal transgene, thus clearing the technical obstacles for building animal models of transgenic diseases except mice.

Application characteristics and prospects of 1.2.2 in TCM research Due to the disunity and uncertainty in theory and method of standardization and quantification of TCM symptoms, the description and representation of TCM symptoms are controversial, which makes the animal models of diseases constructed according to TCM theory very limited. It is helpful for the further development of TCM practice and theory to formulate unified diagnostic criteria of TCM symptoms as soon as possible, explore quantitative methods in line with TCM theory, and explore and reveal the relationship between TCM symptoms and gene expression regulation and their functions. The holistic view and dialectical view of TCM theory emphasize the integrity of the body, the specificity of individuals and the key role of pathogenic dialectics in disease diagnosis, exploration of etiology and pathogenesis and disease treatment, suggesting that it is necessary to understand the etiology and pathogenesis of TCM symptoms with the concepts and methods of functional genomics, transcriptomics, protein genomics and metabonomics. Therefore, from the perspective of genetics, the explanation of TCM symptoms is more reflected in the correlation and functionality (integrity and dialectics) between multiple genes dominated by a certain gene, and more manifested in the occurrence and development of symptoms caused by epigenetic changes (individuality). The great progress of modern gene transformation technology and the emergence of new technologies such as CRISPR/Cas9 make it possible to transform multiple genes in 1 individual economically and quickly, and artificially simulate natural regulation. With the unification and determination of standardization and quantification of TCM symptoms in theory, the continuous improvement and wide recognition of methods, it is believed that the animal model of TCM symptoms constructed by genetic modification technology will come out in the near future, which will provide a new platform for TCM research and is expected to push the practice and theoretical development of TCM to a new height.

2 abstract

Any phenotype, including disease phenotype, can be traced back to the function, regulation and change of related genes. From this point of view, the animal model of disease constructed by gene transformation has grasped the root of the problem of disease occurrence and development, which is helpful for scholars to fundamentally understand the law of disease occurrence and development, and also provides an excellent experimental platform for new, safe and effective drug development. With the deepening understanding of the essence of medicine and genetics, modern gene modification technology represented by CRISPR/Cas9 technology is expected to be more widely used in the construction of animal models of diseases, which will strongly promote the development of modern medicine.

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