Construction strategy and application progress of SARS mouse model
SARS,小鼠模型
z-bo
2020-07-23
712
Although the national epidemic gradually disappeared, the epidemic in Beijing "rebounded" and affected the hearts of people across the country. During active screening, several asymptomatic carriers were found. This must have taught us all: the epidemic is not over yet, we still need to strengthen our epidemic prevention work!
The new global coronavirus (COVID-19) pandemic caused by the new coronavirus (SARS-CoV-2) is a huge challenge for China and the world as a whole. The detailed study of the mechanism of interaction between severe acute respiratory syndrome (SARS) and host molecules, the investigation of the pathogenic process of the virus, the establishment of disease models of SARS virus infection, clinical treatment, vaccines, and drug development are very important as guidance. Whether it is vaccine development, new drug development or gene therapy, accurate animal models are needed to verify the cause of the disease and the immune system.
1. The importance and benefits of using mouse models to study SARS? In the study of human virus infection, the advantages of the mouse model are very obvious. For example, they are cheaper, easier to reproduce, reproduce faster and have more offspring. More importantly, the genetic background of inbred mice is the same. On this basis, mouse genes can be edited and modified to establish the required model, and virus host-specific genes are absolutely useful for research. The impact of disease in the disease process also helps to better understand the host's immune system response and its immune defense capabilities triggered by viruses or vaccines.
Of course, there are restrictions on mouse models. When viruses infect human and mouse cells, they usually bind to specific host cell surface receptors and are related to the host's innate immune response and other factors. In addition, there are species differences between mice and humans. Some human viruses cannot directly infect mice. Alternatively, human viruses can replicate in mice, but it is difficult to cause obvious symptoms of infection. Therefore, be careful when converting the results of mouse model studies to humans. However, the use of different types of gene-edited mice, humanized mice or genetically diverse mouse strains can overcome some of the disadvantages of wild-type mice and make the mouse model a research host, thereby making the virus and pathogenicity interaction. A very useful research tool for evaluating the efficacy, safety and efficacy of vaccines and drugs.
2. What are the strategies and methods for using mouse models to study SARS-CoV?
According to different mouse construction strategies and methods, there are currently three mouse models of SARS-CoV infection:
1. Use human SARS-CoV virus to directly infect inbred mice;
2. Gene mouse technology can be used to edit, knock out mouse-related genes or transfer human host cell SARS virus binding receptors (ACE2, etc.) to mice;
3. Adaptation and evolution of wild-type SARS-CoV virus in mice Repeatedly, virus-adapted and more pathogenic mice are obtained, thereby establishing a virus-infected mouse model that causes a clear clinical phenotype. on
The mouse model of SARS-CoV infection, the main research progress focuses on the following aspects:
(1) Directly use inbred mouse models to study the pathogenicity of SARS virus
SARS-CoV infection causes an increase in mortality and is associated with the increase in severe acute respiratory syndrome (ARDS) in elderly patients (60 years and older). There are various studies to explore better mouse models that can mimic SARS-CoV infection in the elderly. The experiment was performed on BALB/c, C57BL/6 and 129S6 inbred wild mice aged 12 to 14 months. After being infected with SARS-CoV, the three types of old mice had a shorter life span (about 7 days on average) ). Metropolitan) clinical manifestations such as weight loss, messy hair, arching and dehydration, and more importantly, histopathology includes inflammatory cells infiltrating blood vessels and bronchioles, and bronchiolar cell necrosis. , The occurrence of interstitial pneumonia. In addition, the extensive alveolar destruction of BALB/c mice lasted until the 9th day, which was very close to the clinicopathological manifestations of human SARS-CoV infection. This study is also the first example to reveal the genetic and age characteristics of hosts that can significantly affect the pathogenicity of SARS-CoV infection. This also shows that the occurrence of virus infection is related to the characteristics of the host itself and the virus itself. Factors also play a very important role. (2) Using gene editing technology to establish a mouse model of SARS virus infection hACE2 genetically engineered humanized mouse model is currently under construction, with different tissue-specific or extensive expression promoters and hACE2 gene constructs. Usually used. The expression vector is randomly inserted into the transgenic mouse model by prokaryotic injection, such as cytokeratin (epithelial cell specific expression-K18) promoter, CAG promoter (widely expressed), mouse ACE2 gene promoter. The results of studies using these humanized ACE2 mouse models show that the expression level of hACE2 is directly related to the severity of the disease. Although all these mouse models showed infection of respiratory epithelial cells, the high expression of hACE2 gene can also be confirmed in the mouse brain. It was found that the SARS-CoV infection model increased and spread the viral load in the mouse brain. Spread, eventually leading to encephalitis and death of mice.
(3) Obtain SARS-CoV mutant strains using adaptive experimental evolution methods
Mice-specific tissues are planted with viruses in adaptive evolution to adapt the viruses to their specific living environment. Specific selection mutations occur at the bottom to achieve the purpose of more effective replication under pressure. In order to make SARS-CoV replicate in the lungs of wild-type mice and cause severe acute respiratory disease phenotypes, the researchers found that the clinical strain of SARS-CoV has been applied to the nasal cavity of BALB/c mice (6 weeks old). Repeated vaccination 15 times until the mice became wild type showed obvious clinical symptoms of human infection with SARS-CoV (such as weight loss). By analyzing adaptive lethal virus strains obtained from different passages, we analyzed the relationship between severe airway changes caused by the virus and specific mutations in the protein, and analyzed the specific SARS-CoV protein between the virus hosts. Useful for further research on specific mutations. Convenient function connection.
(4) Use CollaboratoryCross-CC technology to establish genetic background diversity mouse strains
Use the CC mouse strain to establish a SARS-CoV infection model. This gene is helpful to find out which related influencing factors in viral diseases determine the host's different response to viral infection. After infecting CC mice with SARS-CoV, we analyzed the CC mice to determine differences in weight loss, virus titer, etc., in order to quickly determine the genes related to the phenotype. Compare with phenotype. Some researchers have used the CC mouse technology to discover CC mouse strains (CC003 and CC053/Unc) that are completely susceptible to the virus during the pathogenic process of SARS-CoV infection. done. In gene mapping analysis, the Toll-like receptor signal transduction pathway aptamer protein Ticam2 is a potentially important element to determine the phenotype of severe respiratory diseases. Based on the results of these studies, CC mice can be used as a new type of mouse model that can more objectively simulate the clinical phenotype of human viral infections. 3. Application progress of mouse model in nucleic acid research of new coronavirus (SARS-CoV-2) By comparing nucleic acid and amino acid sequences, SARS-CoV-2 and SARS-CoV are very similar. (SARS-CoV-2 interacts with the host through the cell surface ACE2 receptor, which is similar (but not exactly the same) as SARS-CoV infection in severe acute respiratory diseases. Animals that cause syndromes and other clinical manifestations, so the above and The strategies and methods for establishing SARS-CoV infection-related mouse models are also suitable for establishing SARS-CoV-2 infection mouse models., by Chinese scientists
is the latest application research of the mouse model in the study of new coronavirus infection.
1. The Qin Q transgenic humanized ACE2 mouse model of the Institute of Medical Experimental Animals, Chinese Academy of Medical Sciences, Dr. Kawa first reported the establishment of a new coronavirus infection hACE2 mouse model: the transgenic hACE2 mouse model is an application mouse, using The mAce2 promoter and the expression vector constructed from the hACE2 gene. We obtained a transgenic hACE2 mouse model by injection: After the hACE2 transgenic mice were infected with the SARS-CoV-2 virus, the mice lost weight and high levels were detected in the lungs. Viral load, infected mice will have moderate interstitial pneumonia, alveolar pathology, such as multiple infiltration of lymphocytes and monocytes in the interstitium, and accumulation of macrophages in the alveolar space. The researchers also used hACE2 transgenic mice for the first time to evaluate the safety and effectiveness of the new coronavirus inactivation. The results showed that different doses and time of inactivated vaccines can immunize hACE2 mice. No inflammation and adverse reactions were observed in the mice. The detection and analysis of the immunogenicity of the inactivated vaccine resulted in the mice producing SARS-CoV-2 specific S protein and RBD specific IgG. The results showed that RBD-specific IgG accounted for 50% of the S protein antibody response, indicating that RBD is the main immunogen of the new coronavirus inactivated vaccine. 2 hACE2 mouse model inserted by artificial fixed points
The research team headed by the Laboratory Animal Resources Institute of China Testing Society reported for the first time that it successfully constructed a hACE2-KI/NIFDC mouse model for fixed point insertion. .. In the humanized mouse construction strategy, the hACE2 gene is directly inserted into the mouse mAce2 gene promoter, and the tdTomato gene is inserted downstream of the hACE2 gene, so that the humanized mouse is under the control of the endogenous mAce2 gene under. , HACE2 and tdTomato genes are co-expressed. Young and old hACE2 mice infected with the SARS-CoV-2 virus in the nasal cavity have higher viral loads in the lungs, trachea and brain of the mice, as well as the spleen, kidney, liver, intestine and serum of the mice. No viral RNA was found. hACE2 mice are both young and old. Interstitial pneumonia is manifested by inflammatory cell infiltration, thickening of alveolar septum and obvious vascular damage. However, the infected mice did not die. Analysis of the main target cells of SARS-CoV-2 infected mice showed that Clara cells, which are positive for Clara cell secretion protein (CC10), are the main target cells of respiratory SARS-CoV-2. The study also confirmed that SARS-CoV-2 intragastric vaccination can also cause hACE2 mouse infection and cause lung pathological changes in hACE2 mice.
3. Mouse model produced by adenovirus vector
Professor Zhao Jincun's team from Guangzhou National Institute of Respiratory Diseases used adenoviral vectors (Ad5) to transduce and express hACE2 in mouse lungs and successfully established the world's first non-transgenic mouse model of novel coronavirus pneumonia. The adenovirus (Ad5) vector (Ad5-CMV-hACE2) expressing the hACE2 gene was introduced via the CMV promoter, and it was introduced intranasally into wild mice and related gene knockout mice (type I interferon). Based on receptor-deficient or STAT1 knockout mice, a mouse model of mouse lung expressing hACE2 gene has been established, which is a key gene in the interferon pathway. When this non-transgenic humanized hACE2 mouse was infected with SARS-CoV-2, a high titer of the virus was detected in the lungs of the mouse, resulting in weight loss and clinicopathological symptoms and patients with new coronary pneumonia similar. Will appear. At the same time, experiments in mouse models also proved that type I interferon and STAT1 genes may be protected by SARS-CoV-2 infection. As a leader in the development of model animals,
Zengyo Bio formed a research and development team to develop a new coronary mouse model at the beginning of the new coronavirus epidemic. We use the in-house developed TurboKnockout technology and optimized CRISPR-Pro technology to simultaneously prepare humanized hACE2 mice from three background strains of BALB/c, C57BL/6J and C57BL/6N to fully explore the advantages of different gene editing methods . Disadvantages, different gene targeting programs and construction strategies are designed to meet the needs of different research purposes and different application directions. Half a year has passed since the outbreak of new coronary pneumonia, and the research and development of vaccines and antiviral drugs are gradually becoming obvious. Although the staged victory of an epidemic is not immediately useful for preventing or treating infected people, everyone knows it. In this process, the mouse has become a powerful assistant in the human anti-viral infection pathway, realizing its greatest value to humans. Human beings have a common destiny and living things live together.
