Genetically engineered mice are important tools for elucidating gene function and disease mechanisms and for advancing drug development. With the growing demand in biomedical research for complex genetic modifications, rapid model generation, and high-throughput functional analysis, technologies for generating genetically engineered mice continue to evolve. This review systematically summarizes the evolution and applications of these technologies, with a focus on conventional approaches, including early transgenic technologies and embryonic stem cell (ESC)-based homologous recombination for gene targeting, as well as the use of random mutagenesis approaches, such as N-ethyl-N-nitrosourea (ENU) mutagenesis and transposon insertion, in forward genetic screening. It further discusses the principles and characteristics of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas systems, as well as their applications in generating genetically engineered mice. With advances in genome editing, Cas9-derived systems, base editing, prime editing, and RNA editing further improve editing precision, expand the repertoire of genetic modifications and precise regulatory approaches, and provide additional options for generating genetically engineered mice. This review also compares the advantages, limitations, and suitability of different technologies for specific applications and summarizes quality-control strategies addressing practical issues that may affect model reliability, including off-target effects, mosaicism, genetic background, and genetic stability. Finally, future directions in technologies for generating genetically engineered mice are discussed to inform the generation and application of related models.

Peanut, a major economic and oil crop known for the high protein and oil content, is extensively cultivated in China. Peanut plants have the ability to form nodules with rhizobia, where the nitrogenase converts atmospheric nitrogen into ammonia nitrogen that can be utilized by the plants. Analysis of nodule fixation is of positive significance for avoiding overapplication of chemical fertilizer and developing sustainable agriculture. In this study, AhNIGT1.2, a member of the NIGT family predominantly expressed in peanut nodules, was identified by bioinformatics analysis. Subsequent spatiotemporal expression analysis revealed that AhNIGT1.2 was highly expressed in nodules and showed significant responses to high nitrogen, low nitrogen, high phosphorus, low phosphorus, and rhizobia treatments. Histochemical staining indicated that the gene was primarily expressed in developing nodules and at the connection region between mature nodules and peanut roots. The fusion protein AhNIGT1.2-GFP was located in the nucleus of tobacco epidermal cells. The AhNIGT1.2-OE significantly increased the number of peanut nodules, while AhNIGT1.2-RNAi reduced the number of nodules, which suggested a positive regulatory role of AhNIGT1.2 in peanut nodulation. The AhNIGT1.2-OE in roots down-regulated the expression levels of NRT1.2, NRT2.4, NLP1, and NLP7, which indicated that AhNIGT1.2 influenced peanut nodulation by modulating nitrate transport and the expression of NLP genes. The transcriptome analysis of AhNIGT1.2-OE and control roots revealed that overexpressing AhNIGT1.2 significantly enriched the differentially expressed genes associated with nitrate response, nodulation factor pathway, enzymes for triterpene biosynthesis, and carotenoid biosynthesis. These findings suggest that AhNIGT1.2 play a key role in peanut nodulation by regulating nitrate transport and response and other related pathways. This study gives insights into the molecular mechanisms of nitrogen and phosphorus in regulating legume nodulation and nitrogen fixation, and sheds light on the development of legume crops that can efficiently fix nitrogen in high nitrogen environments.
Arachis/physiology* ; Nitrates/metabolism* ; Plant Proteins/physiology* ; Transcription Factors/metabolism* ; Plant Root Nodulation/physiology* ; Gene Expression Regulation, Plant ; Root Nodules, Plant/metabolism* ; Nitrogen Fixation

Objective To explore the efficacy and underlying mechanism of Astragalus in the treatment of viral pancreatitis using network pharmacology,with confirmation of its efficacy and mechanism in cell experiments.Methods Astragalus and viral pancreatitis targets obtained from the Traditional Chinese Medicine Systems Pharmacology(TCMSP)and GeneCards databases were combined to obtain intersection targets.GO functional enrichment and KEGG signaling pathway enrichment analyses of therapeutic targets were conducted using the Database for Annotation,Visualization,and Integrated Discovery(DAVID)database.The interactions between therapeutic targets were analyzed using the STRING database and Cytoscape 3.10.2,and the core therapeutic targets were screened.Molecular docking between the most effective therapeutic components and the core targets was performed using PyMOL 3.0 and AutoDock Tools 1.5.7.CVB3 was used to construct a viral pancreatitis cell model for verification of the core targets.Results Seventy-eight therapeutic targets were identified.Enrichment analyses revealed the possible involvement of pathways related to cancer,lipids and atherosclerosis,and PI3K-AKT signaling.AKT1,TP53,HIF1A,CASP3,IL-6,and MMP9 were identified as possible core targets.The result of cell experiments showed that the expression level of AMY was significantly increased in the model group(P＜0.05).The Astragalus injection group exhibited significantly decreased expression levels of AMY,AKT1,TP53,HIF1A,CASP3,IL-6,and MMP9 compared with the model group(P＜0.05).Conclusions Astragalus injection effectively treated viral pancreatitis,and its therapeutic mechanism may involve reduced expression levels of AKT1,TP53,HIF1A,CASP3,IL-6,and MMP9.

Objective To establish an animal model of hand,foot,and mouth disease(HFMD)in Syrian hamsters coinfected with coxsackievirus A6(CVA6)and coxsackievirus B1(CVB1).Methods 42 Syrian hamsters were divided into a CVA6 infection group,CVB1 infection group,CVA6 and CVB1 coinfection group and control group.A HFMD model was established by nasal instillation of virus solution and phosphate-buffered saline.Clinical and physiological indicators and detoxification status were monitored and recorded for 15 d,and animals were selected on day 7(D7)after infection for histopathology and viral antigen and nucleic acid testing.Results Hamsters in the single-infection and coinfection groups showed clinical symptoms similar to human HFMD.White blood cell,neutrophil,and lymphocyte result were characteristic of viral infection.Both viral nucleic acids were detected in throat swabs,feces,blood,and tissues and both viruses were isolated from fecal samples.Pathological damage and positive co-localization of CVA6 and CVB1 viral antigen proteins and nucleic acids were found in brain and other tissues.Conclusions Nasal instillation of a CVA6 and CVB1 mixture can successfully coinfect Syrian hamsters,replicate herpes infection similar to human HFMD,and cause pathological viral myocarditis and encephalitis damage.The result showed that the coinfection group was more seriously affected than the single-infection group,with worse clinical symptoms,increased viral replication,and obvious tissue pathological damage.This study provides a reference for further basic and clinical research into human enterovirus coinfection.

Objective Based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses(PRISMA)guidelines,to obtain precise and reliable comprehensive effect conclusions by quantitatively combining pharmacodynamic results from animal experiments investigating Astragali Radix(single-entity Astragali Radix preparation)or its ingredients for treatment of acute pancreatitis(AP)in literature reports through meta-analysis.Methods Databases including China National Knowledge Infrastructure(CNKI),VIP Database for Chinese Technical Periodicals(VIP),Wanfang Data Knowledge Service Platform,China Biomedical Literature Database(CBMdisc),PubMed,and Web of Science(WOS)were searched from inception to March 2025 for animal studies related to Astragali Radix(single-entity Astragali Radix preparation)or its ingredients for AP treatment.Risk of bias for included studies was assessed with SYRCLE tool.Heterogeneity among studies was evaluated according to Cochrane Handbook using Cochrane's Qtest and/2statistic.Sensitivity analysis was performed using the leave-one-out method,and publication bias risk was detected using Egger's test.Results A total of 297 articles were retrieved,and after screening and evaluation,19 animal studies were finally included for meta-analysis.These 19 publications cover SD rats,as well as three breeds of mice:C57BL/6 mice,BALB/c mice,and Kunming mice.SYRCLE scores ranged from 3 to 4.The results of the sensitivity analysis showed that no study significantly affected the heterogeneity index.The results of Egger's test showed a significant publication bias with P＜0.05.Cochrane's Qtest and I2statistic indicated substantial heterogeneity among studies.Meta-analysis results of 19 animal studies showed that single-entity Astragali Radix preparation(Astragali Radix injection)could reduce serum amylase(AMY)levels,an AP-specific indicator.The Astragali Radix ingredients could decrease both AMY and lipase(LPS)levels.Astragali Radix injection or its ingredients could reduce serum levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-1 β,while increasing IL-10 levels;could increase serum levels of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),and decrease malondialdehyde(MDA)levels.High-dose groups of Astragali Radix injection or Astragali Radix ingredients were more effective than low-dose groups in reducing AMY,TNF-α,and IL-6 levels and increasing SOD levels,but dosage effect on MDA levels was not demonstrated.Conclusion Evidence-based analysis of animal experiment results shows that in various animal models including SD rats,C57BL/6 mice,BALB/c mice,and Kunming mice,Astragali Radix injection or its ingredients can effectively reduce expression or secretion levels of AP-specific indicators(AMY and LPS).The mechanisms may be related to some inflammatory mediators,including reducing TNF-α,IL-6,and IL-1 β levels and increasing IL-10 levels;They may also intervene in oxidative/antioxidative equilibrium,such as increasing SOD and GSH-Px levels and reducing MDA levels.Except for MDA,dose-response relationships are shown for reducing AMY,TNF-α,and IL-6 levels and increasing SOD levels with Astragali Radix injection or its ingredients.However,due to high heterogeneity,potential publication bias risk,and species differences between animal models and human diseases in existing studies,further high-quality clinical trials or animal experiments are still needed in the future.

Objective Based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses(PRISMA)guidelines,to obtain precise and reliable comprehensive effect conclusions by quantitatively combining pharmacodynamic results from animal experiments investigating Astragali Radix(single-entity Astragali Radix preparation)or its ingredients for treatment of acute pancreatitis(AP)in literature reports through meta-analysis.Methods Databases including China National Knowledge Infrastructure(CNKI),VIP Database for Chinese Technical Periodicals(VIP),Wanfang Data Knowledge Service Platform,China Biomedical Literature Database(CBMdisc),PubMed,and Web of Science(WOS)were searched from inception to March 2025 for animal studies related to Astragali Radix(single-entity Astragali Radix preparation)or its ingredients for AP treatment.Risk of bias for included studies was assessed with SYRCLE tool.Heterogeneity among studies was evaluated according to Cochrane Handbook using Cochrane's Qtest and/2statistic.Sensitivity analysis was performed using the leave-one-out method,and publication bias risk was detected using Egger's test.Results A total of 297 articles were retrieved,and after screening and evaluation,19 animal studies were finally included for meta-analysis.These 19 publications cover SD rats,as well as three breeds of mice:C57BL/6 mice,BALB/c mice,and Kunming mice.SYRCLE scores ranged from 3 to 4.The results of the sensitivity analysis showed that no study significantly affected the heterogeneity index.The results of Egger's test showed a significant publication bias with P＜0.05.Cochrane's Qtest and I2statistic indicated substantial heterogeneity among studies.Meta-analysis results of 19 animal studies showed that single-entity Astragali Radix preparation(Astragali Radix injection)could reduce serum amylase(AMY)levels,an AP-specific indicator.The Astragali Radix ingredients could decrease both AMY and lipase(LPS)levels.Astragali Radix injection or its ingredients could reduce serum levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-1 β,while increasing IL-10 levels;could increase serum levels of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),and decrease malondialdehyde(MDA)levels.High-dose groups of Astragali Radix injection or Astragali Radix ingredients were more effective than low-dose groups in reducing AMY,TNF-α,and IL-6 levels and increasing SOD levels,but dosage effect on MDA levels was not demonstrated.Conclusion Evidence-based analysis of animal experiment results shows that in various animal models including SD rats,C57BL/6 mice,BALB/c mice,and Kunming mice,Astragali Radix injection or its ingredients can effectively reduce expression or secretion levels of AP-specific indicators(AMY and LPS).The mechanisms may be related to some inflammatory mediators,including reducing TNF-α,IL-6,and IL-1 β levels and increasing IL-10 levels;They may also intervene in oxidative/antioxidative equilibrium,such as increasing SOD and GSH-Px levels and reducing MDA levels.Except for MDA,dose-response relationships are shown for reducing AMY,TNF-α,and IL-6 levels and increasing SOD levels with Astragali Radix injection or its ingredients.However,due to high heterogeneity,potential publication bias risk,and species differences between animal models and human diseases in existing studies,further high-quality clinical trials or animal experiments are still needed in the future.

Objective To establish an animal model of hand,foot,and mouth disease(HFMD)in Syrian hamsters coinfected with coxsackievirus A6(CVA6)and coxsackievirus B1(CVB1).Methods 42 Syrian hamsters were divided into a CVA6 infection group,CVB1 infection group,CVA6 and CVB1 coinfection group and control group.A HFMD model was established by nasal instillation of virus solution and phosphate-buffered saline.Clinical and physiological indicators and detoxification status were monitored and recorded for 15 d,and animals were selected on day 7(D7)after infection for histopathology and viral antigen and nucleic acid testing.Results Hamsters in the single-infection and coinfection groups showed clinical symptoms similar to human HFMD.White blood cell,neutrophil,and lymphocyte result were characteristic of viral infection.Both viral nucleic acids were detected in throat swabs,feces,blood,and tissues and both viruses were isolated from fecal samples.Pathological damage and positive co-localization of CVA6 and CVB1 viral antigen proteins and nucleic acids were found in brain and other tissues.Conclusions Nasal instillation of a CVA6 and CVB1 mixture can successfully coinfect Syrian hamsters,replicate herpes infection similar to human HFMD,and cause pathological viral myocarditis and encephalitis damage.The result showed that the coinfection group was more seriously affected than the single-infection group,with worse clinical symptoms,increased viral replication,and obvious tissue pathological damage.This study provides a reference for further basic and clinical research into human enterovirus coinfection.

Objective To explore the efficacy and underlying mechanism of Astragalus in the treatment of viral pancreatitis using network pharmacology,with confirmation of its efficacy and mechanism in cell experiments.Methods Astragalus and viral pancreatitis targets obtained from the Traditional Chinese Medicine Systems Pharmacology(TCMSP)and GeneCards databases were combined to obtain intersection targets.GO functional enrichment and KEGG signaling pathway enrichment analyses of therapeutic targets were conducted using the Database for Annotation,Visualization,and Integrated Discovery(DAVID)database.The interactions between therapeutic targets were analyzed using the STRING database and Cytoscape 3.10.2,and the core therapeutic targets were screened.Molecular docking between the most effective therapeutic components and the core targets was performed using PyMOL 3.0 and AutoDock Tools 1.5.7.CVB3 was used to construct a viral pancreatitis cell model for verification of the core targets.Results Seventy-eight therapeutic targets were identified.Enrichment analyses revealed the possible involvement of pathways related to cancer,lipids and atherosclerosis,and PI3K-AKT signaling.AKT1,TP53,HIF1A,CASP3,IL-6,and MMP9 were identified as possible core targets.The result of cell experiments showed that the expression level of AMY was significantly increased in the model group(P＜0.05).The Astragalus injection group exhibited significantly decreased expression levels of AMY,AKT1,TP53,HIF1A,CASP3,IL-6,and MMP9 compared with the model group(P＜0.05).Conclusions Astragalus injection effectively treated viral pancreatitis,and its therapeutic mechanism may involve reduced expression levels of AKT1,TP53,HIF1A,CASP3,IL-6,and MMP9.

Objective:To evaluate the application of the standard of "Delimitation and Classification of Keshan Disease Areas" (GB 17020-2010, Standard for short), learn about the applicability of its technical indicators and requirements, and provide a basis for revision of the Standard.Methods:In March 2022, provinces with severe epidemic areas of Keshan disease or new cases reported in recent years were selected, including Gansu Province, Inner Mongolia Autonomous Region, Shandong Province, Shaanxi Province, Yunnan Province, Shanxi Province, Liaoning Province, and Chongqing City. Multi-stage stratified sampling method and questionnaire survey were adopted to collect information on the application of Standard by relevant health institutions.Results:A total of 448 questionnaires were collected, including 445 valid. The survey results showed that 87.64% (390/445) of the respondents were aware of the Standard, and 64.72% (288/445) had received training on the Standard. Eighty-two point two per cent (365/445) of the respondents believed that the Standard was simple and easy to operate, 83.82% (373/445) believed that the determination of the diseased townships was scientific and reasonable, and could be effectively implemented, and 83.60% (372/445) believed that the determination of historical epidemic areas was applicable to the current situation of Keshan disease; 38.88% (173/445), 38.20% (170/445), and 37.98% (169/445) of the respondents believed that the classification indicators for classifying epidemic areas (severe, moderate, and mild epidemic areas) were not applicable to the current situation of the disease in the local or provinces with Keshan disease, respectively. Among the indicators for epidemic areas classification, 30.79% (137/445), 29.21% (130/445), and 28.54% (127/445) of the respondents thought that the annual prevalence, the annual number of new cases and the annual incidence were most suitable for classification of Keshan disease areas.Conclusions:The Standard has been applied well in practice. However, based on the current situation of Keshan disease, it is suggested to redefine the standard for the severity of the disease and the classification of historical epidemic areas.

Objective:To study the correlation between serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) level and echocardiographic parameters in patients with chronic Keshan disease, providing reference for diagnosis and prognosis evaluation of chronic Keshan disease.Methods:Ninety-nine patients with chronic Keshan disease who received standardized treatment at Jingchuan County People's Hospital in Pingliang City, Gansu Province from January to December 2020 were selected. Among them, 16 patients were classified as cardiac function grade Ⅱ according to New York Heart Association (NYHA), 69 as grade Ⅲ and 14 as grade Ⅳ. The patients underwent echocardiography and their serum NT-proBNP level was measured using fluorescence immunochromatography. The differences in serum NT-proBNP levels among patients with different cardiac function grades were compared, and the correlation between cardiac function grades, serum NT-proBNP level and echocardiographic parameters was analyzed.Results:The serum NT-proBNP levels in patients with cardiac function grades Ⅱ, Ⅲ, and Ⅳ were (1 107.26 ± 268.03), (2 125.98 ± 293.02), and (8 268.59 ± 2 659.50) pg/ml, respectively. The differences among the three groups were statistically significant ( F = 13.94, P < 0.001). The serum NT-proBNP level was positively correlated with cardiac function grades ( r = 0.44, P < 0.001), left ventricular end-diastolic diameter, left ventricular end-systolic diameter, and left atrial diameter ( r = 0.45, 0.52, 0.38, P < 0.001), and negatively correlated with fractional shortening and left ventricular ejection fraction ( r = - 0.39, - 0.46, P < 0.001). Conclusions:The serum NT-proBNP level in patients with chronic Keshan disease with different cardiac function grades is different, and is positively correlated with echocardiographic parameters reflecting the degree of cardiac structural and functional impairment. The NT-proBNP level may become an early diagnostic, grading, and prognostic indicator for chronic Keshan disease.