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China Agricultural University

17 ARTICLES PUBLISHED IN JoVE

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Medicine

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor
Jae H.T. Lee 1, Femke Streijger 1, Seth Tigchelaar 1, Michael Maloon 1, Jie Liu 1, Wolfram Tetzlaff 1, Brian K. Kwon 1,2
1International Collaboration on Repair Discoveries (ICORD), University of British Columbia , 2Department of Orthopaedics, University of British Columbia

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

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Medicine

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration
Jianhua Xiong 1, Jian Hou 2
1Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health, 2State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

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Developmental Biology

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro
Zhaofeng Jia 1,2, Yujie Liang 3, Bin Ma 4,5, Xiao Xu 2,6, Jianyi Xiong 2, Li Duan 2, Daping Wang 1,2
1Guangzhou Medical University, 2Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopeadic Engineering, Department of Orthopedics, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), 3Department of Chemistry, The Chinese University of Hong Kong, 4School of Biomedical Engineering, Shanghai Jiao Tong University, 5Renji Hospital Clinical Stem Cell Research Center, Shanghai Jiao Tong University School of Medicine, 6Shantou University Medical College

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

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Neuroscience

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization
Xiao Xu 1, Yinwei You 1,2, Long Zhang 1
1Department of Entomology, China Agricultural University, 2Bio-tech Research Center, Shandong Academy of Agricultural Sciences

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

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Medicine

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo
Mingming Liu 1, Xiaoyan Zhang 1, Bingwei Li 1, Bing Wang 1, Qingbin Wu 1, Fei Shang 1, Ailing Li 1, Hongwei Li 1, Ruijuan Xiu 1
1Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet β cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

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Biology

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model
Zhaofeng Jia 1,2,3, Yujie Liang 4,5, Xingfu Li 2,3, Xiao Xu 1,2,3, Jianyi Xiong 2,3, Daping Wang 1,2,3, Li Duan 2,3
1Postgraduate institution, Guangzhou Medical University, 2Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, 3Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopaedic Engineering, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), 4Department of Chemistry, Chinese University of Hong Kong, 5Shenzhen Kangning Hospital, Shenzhen Mental Health Center

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

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Environment

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea
Mengyu Zhang *1, Xuan Sun *1, Lie Cui 1, Yue Yin 1, Xinyu Zhao 1, Song Pan 2, Weixiang Wang 1
1Beijing Key Laboratory of New Technology in Agricultural Application, National Demonstration Center for Experimental Plant Production Education, Beijing University of Agriculture, 2College of Plant Protection, State Key Laboratory of Agrobiotechnology, Ministry of Agriculture Key Laboratory of Pest Monitoring and Green Management, China Agricultural University

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

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Neuroscience

Single Sensillum Recordings for Locust Palp Sensilla Basiconica
Hongwei Li 1, Yinwei You 2, Long Zhang 1
1Department of Entomology, China Agricultural University, 2Bio-tech Research Center, Shandong Academy of Agricultural Sciences

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

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Immunology and Infection

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells
Yi Lu 1,2,3, Xin-Yuan Fu 3, Yongliang Zhang 1,2
1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

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Immunology and Infection

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice
Yongli Zhang *1,2, Hangjie Zhang *2, Wenqiang Ma *3, Kefang Liu 1,2, Min Zhao 4, Yingze Zhao 2, Xuancheng Lu 5, Fuping Zhang 6, Xiangdong Li 3, George F. Gao 1,2,4,6, William J. Liu 1,2
1School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, 2NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 3State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, 4Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, 5Laboratory Animal Center, Chinese Center for Disease Control and Prevention, 6CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

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Medicine

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy
Wen-Hui Wang 1, Long Zhang 2,3, Guo-Xiang Dong 2, Jun Zhao 2, Xuan Li 2,3
1Department of Dermatology, Peking University Third Hospital, 2Department of Interventional Radiology and Vascular Surgery, Peking University Third Hospital, 3Wound Healing Center, Peking University Third Hospital

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

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Biology

Simple Homemade Tools to Handle Fruit Flies—Drosophila melanogaster
Daxiang Yang 1
1Department of Zoology and Animal Physiology, College of Biological Sciences, China Agricultural University

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

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Biology

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites
Chunhui Duan 1, Xinming Tang 1, Dandan Hu 1, Sixin Zhang 1, Jie Liu 1, Feifei Bi 1, Zhenkai Hao 1, Jingxia Suo 1, Ying Yu 1, Min Wang 1, Pei Sun 1, Liying Du 2, Xun Suo 1, Xianyong Liu 1
1National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, 2School of Life Sciences, Peking University

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

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Immunology and Infection

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks
Yongliang Zhang *1, Yuanyuan Li *2,3, Xinxin Yang 1, Zhiyan Wen 1, Ugrappa Nagalakshmi 2, Savithramma P. Dinesh-Kumar 2,3
1State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, 2Department of Plant Biology, College of Biological Sciences, University of California, Davis, 3Genome Center, College of Biological Sciences, University of California, Davis

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

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Biology

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture
Li Duan 1,2, Xingfu Li 1,2, Xiao Xu 1,2, Limei Xu 1,2, Daping Wang 1,2, Kan Ouyang 1,2, Yujie Liang 1,3
1Department of Orthopedics, The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital, 2Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, Shenzhen Second People's Hospital, 3Department of Child and Adolescent Psychiatry, Shenzhen Kangning Hospital, Shenzhen Mental Health Center, Shenzhen Key Laboratory for Psychological Healthcare & Shenzhen Institute of Mental Health

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

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Behavior

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults
Tong Li *1,2, Yujia Weng *1,2, Daxiang Yang 1
1Department of Zoology and Animal Physiology, College of Biological Sciences, China Agricultural University, 2College of Life Sciences, Capital Normal University

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

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Immunology and Infection

Examination of Pyroptosis by Flow Cytometry
He Chang 1,2,3, Zhi Chen 1,2,3, Li Gao 1,2,3, Hong Cao 1,2,3, Yongqiang Wang 1,2,3, Shijun J. Zheng 1,2,3
1National Key Laboratory of Veterinary Public Health Security, 2Animal Epidemiology of the Ministry of Agriculture, 3College of Veterinary Medicine, China Agricultural University

This article describes the identification of pyroptotic cells using flow cytometry after dual staining with antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI).

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