New crown mouse animal model helps fight the epidemic
The global research and development of the new global coronavirus vaccine has made considerable progress, and vaccines prepared through various technical methods have entered phase II clinical trials. However, the relative lack of animal models has had a significant impact on the development and clinical trials of the new crown vaccine at this stage. The selection, preparation and application of animal models need to be further improved to improve vaccines or antiviral drugs. The steps of drug development and manufacturing ensure the safety and quality of human life.
Overview of mouse models in SARS research
History is always surprisingly similar. From SARS to MERS, the animal models used have important reference significance for choosing new coronavirus animal models. I will. In 2016, Professor Troy C Sutton and Professor Kanta Subbarao from the Institute of Infectious Diseases of the National Institutes of Health published a review article detailing the development and application of animal models in coronavirus research. From the mouse model, the researchers conducted challenging experiments by using BALB/c inbred mice to inject SARS-CoV from the nasal cavity. As a result, the mice lost weight and reduced lung diseases. Have not experienced clinical phenomena. C57BL/6 mice obtained similar results, but still lacked a clinical phenotype. Under the background conditions of B6 and 129SvEv, CD1-/- mice lacking NK cell function or NK-T cells have a virus replication rate similar to RAG1-/- mice lacking T and B lymphocytes. The phenotype of the clinical disease has not been shown. Histopathological examination of STAT1-/- mice on B6 background showed self-limiting bronchiolitis and lamellar interstitial pneumonia. However, 1291-SvEv background STAT1-/- mice showed progressive weight loss and bronchiolitis, which developed into interstitial pneumonia and mediastinitis. Although the mice showed signs of infection and lung disease, the inbred mice failed to accurately reproduce the diffuse alveolar damage, edema, lung cell necrosis, and hyalin formation observed in humans. In order to simulate the increased mortality associated with aging in epidemiology, the researchers developed an older SARS-CoV mouse model. In this model, the level of virus replication in the lungs of BALB/c and B6 mice was high, both mice lost weight, and adult BALB/c mice also exhibited fur folds and dehydration. In all experiments, older mouse models are more widely used than older mouse models because the response of older mouse models to viruses is similar to the viral infection phenotype displayed by humans. In order to establish a lethal mouse model of SARS-CoV infection, researchers have developed transgenic mice expressing human ACE2 (hACE2). Generally, the severity of transgenic mice is related to the expression level of hACE2. Transgenic mice expressing hACE2 under the control of the cytokeratin promoter have high levels of ACE2 mRNA in the lung, liver, colon, and kidney. When these mice were exposed to SARS-CoV, the infection started in the respiratory epithelium and gradually spread to the brain, and the death rate reached 100% by day 7. Another ACE2 model expresses hACE2 under the control of the chicken β-actin promoter of the cytomegalovirus IE enhancer, resulting in transgenic mouse strains with different levels of hACE2. SARS-CoV-infected mice with high expression of hACE2 caused severe lung and brain infections, with a mortality rate of 100%. Such mice exhibit severe interstitial pneumonia, causing damage to organs outside the lungs, so that the pathology of human SARS-CoV infection can be simulated relatively accurately. The above studies indicate that the expression of hACE2 under the control of the mouse ACE2 promoter may cause the tissue distribution of hACE2 to be restricted. SARS-CoV challenged hACE2 low-expressing mice more sleepy, but did not suffer serious infection and death. Studies have also shown that an increase in viral load or viral antigens is observed in the brain tissue of transgenic mice, and that the widespread spread of the virus in the brain leads to an increase in mortality, while human central nervous system infections I found almost never observed it. Therefore, although transgenic mice provide a lethal model of SARS-CoV infection, no mouse model can replicate the entire process of SARS-CoV infected virus patients in a complete and accurate manner. Continuously exploring and verifying
In the process of SARS-CoV mouse model, it was discovered that gene editing technology is an important limiting factor in the development of mouse models. With the discovery and spread of CRISPR/Cas9, humanized mouse models emerged. The model can insert and express genes at a specific time and location, more accurately simulate the state of human infection, and have a clearer understanding of the methods and processes of virus-human interaction. We can prepare a more reasonable solution to accommodate all kinds of unfriendly "foreign guests".
The new coronavirus is an unwelcome guest. It is very similar to SARS-CoV, so the above-mentioned mouse model is very important for studying the new coronavirus. Based on these studies, the researchers used new technologies to update and improve their models, and build different types of new coronary mouse models. So how are these new crown mouse models made? Is it possible to accurately and comprehensively simulate the human virus infection process?
Comparison and analysis of various new crown mouse models
The basis for the development of the new crown mouse model is gene editing technology. So, do we need to further study how to prepare the new crown mouse model born under the current gene editing technology? Where are these applications worth?
On June 5, 2020, the Beijing Institute of Microbiology and Epidemiology, the National Institute of Pathogenic Microbiology and Epidemiology, and the National Institute published an article titled "SARS-CoV-2 infection and "The mouse model of pathogenesis" paper and conducted research. Through the new coronavirus attack experiment, the new hACE2 transgenic mouse model has successfully simulated the human body's viral etiology, pathological characteristics and infection methods.
The “targeting strategy” is to insert the complete hACE2 cDNA into the second exon of the mACE2 gene at the GRCm38.p6 locus on the X chromosome to disrupt the expression of mACE2 and terminate it. The tdTomato gene was inserted downstream of hACE2 with an internal ribosome entry site (IRES) so that hACE2 and tdTomato could be co-expressed. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and polyA sequence were added to improve the stability and translation efficiency of mRNA. This targeting strategy allows hACE2 expression to be under the control of the mACE2 promoter. The fertilized eggs of C57BL/6 mice were injected with targeting constructs, subgenomic RNA (sgRNA) and Cas9 mRNA.
This mouse model was created using CRISPR/Cas9 knock-in technology, replacing endogenous mouse ACE2 (mACE2) with human ACE2 (hACE2) earlier than wild-type C57BL/6 mice. After intranasal infection, mice maintain high viral loads in the lungs, trachea, and brain. No deaths were observed, but interstitial pneumonia and elevated cytokines were found in hACE2 mice infected with the new coronavirus. Interestingly, the study also found that inoculating the new coronavirus into the stomach of hACE2 mice may cause infection and cause lung pathological changes. This will bring new ideas about the infection and spread of the new coronavirus. In addition to the new transgenic method, there is also a method to prepare new coronary artery model mice by transducing hACE2 with adenovirus vectors. On June 10, 2020, the team of Professor Zhao Jincun of the Guangzhou National Key Research Institute of Respiratory Diseases and multiple departments jointly announced a new coronavirus mouse model created using adenovirus vectors. .. As shown in Figure 4, in this study, the replication-deficient adenovirus (Ad5-hACE2) exogenous hACE2 was delivered intranasally to C57BL/6 and BALB/c strains and the corresponding knockout mice. And carried out western blotting and flow cytometry. Challenge to detect hACE2 expression by cytometer. In this study, it was observed that hACE2 is mainly expressed in alveolar epithelium, and there may be positive cells in respiratory epithelium. C57BL/6 and new coronaviruses infected with Ad5-hACE2 transduced BALB/c mice showed symptoms such as wrinkles, breathing difficulties, and dyspnea 2 days after infection, similar to SARS-CoV infected BALB/c mice mouse. Start showing. The lung tissues of both mouse strains showed multiple injuries, including perivascular to interstitial inflammatory cell infiltration, necrotic cell fragments, and alveolar edema. All this shows that the new coronavirus mouse model created in this way is susceptible to new coronaviruses and can effectively evaluate the efficacy of vaccines and drugs. The research team at the University of Washington School of Medicine used AdV-hACE2 to transduce BALB/c mice intranasally and intravenously, and used the same method to prepare a new coronary mouse model. The results showed that the adenovirus transduction of hACE2 caused SARS-CoV-2 to infect BALB/c mice, which could produce highly effective neutralizing antibodies. As a leader in the development of model animals,
Zengyo Bio formed a research and development team to develop a new coronavirus mouse model in the early stage of the new coronavirus epidemic. We use the TurboKnockout technology developed by ourselves and the optimized CRISPR-Pro technology to simultaneously prepare humanized mice of three background strains of BALB/c, C57BL/6J, C57BL/6N and three background strains. We comprehensively consider various genes With the advantages and disadvantages of editing methods, various gene targeting programs have been designed to meet the needs of different research purposes and different application directions.
Six months have passed since the new crown epidemic, and the development of vaccines and antiviral drugs is gradually becoming apparent. It cannot be used immediately to prevent or treat infected people, but the victory of the gradual epidemic is obvious to everyone. In this process, the mouse has become a powerful helper on the road of human popularity, and has realized its greatest value to mankind. Human beings have a common destiny and living things live together.
动物模型,新冠小鼠
2020-07-22
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