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Chinese Academy of Medical Sciences

22 ARTICLES PUBLISHED IN JoVE

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Developmental Biology

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors
Wei Wen 1, Jian-Ping Zhang 1, Wanqiu Chen 2, Cameron Arakaki 2, Xiaolan Li 1,2, David Baylink 2, Gary D. Botimer 3, Jing Xu 1, Weiping Yuan 1, Tao Cheng 1,4,5,6,7, Xiao-Bing Zhang 1,2
1State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 2Division of Regenerative Medicine, Department of Medicine, Loma Linda University, 3Department of Orthopaedic Surgery, Loma Linda University, 4Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, 5Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, 6Collaborative Innovation Center for Cancer Medicine, 7Tianjin Key Laboratory of Blood Cell Therapy and Technology

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

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Bioengineering

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex
Rong Li *1, Jinxin Miao *1,2, Zhiqiang Fan 1, SeokHwan Song 3, Il-keun Kong 3,4, Yaohe Wang 2,5, Zhongde Wang 1
1Department of Animal, Dairy, and Veterinary Sciences, Utah State University, 2National Centre for International Research in Cell and Gene Therapy, Sino-British Research Centre, School of Basic Medical Sciences, Zhengzhou University, 3Department of Animal Science Division of Applied Life Science (BK21 Plus), Gyeongsang National University, 4Institute of Agriculture and Life Science, Gyeongsang National University, 5Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

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Biochemistry

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers
Huijuan You *1, Shimin Le *2,3, Hu Chen 2,4, Linyan Qin 1, Jie Yan 2,4,5
1School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 2Mechanobiology Institute, National University of Singapore, 3Department of Physics, National University of Singapore, 4Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics, Xiamen University, 5Center for Bioimaging Sciences, National University of Singapore

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

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Cancer Research

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Xiaoxing Lv 1, Meiru Zhao 1, Yuting Yi 1, Lucheng Zhang 1, Yanfang Guan 1, Tao Liu 1, Ling Yang 1, Rongrong Chen 1, Jianhui Ma 1, Xin Yi 1
1Geneplus-Beijing Institute

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

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Immunology and Infection

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection
Chongfeng Xu 1, Junhong Ai 2, Qingxun Zhang 1, Ting Li 3, Xiaorong Wu 1, Zhengde Xie 2, Ziyuan Duan 1
1Genetic Resources Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 2MOE Key Laboratory of Major Diseases in Children, National Key Discipline of Pediatrics, National Clinical Research Center for Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, 3State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

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Bioengineering

Visual Detection of Multiple Nucleic Acids in a Capillary Array
Jianwei Chen *1,2,3, Ning Shao *1,2,3, Jiaying Hu *4, Rong Li 4, Yuanshou Zhu 1,2,3, Dabing Zhang 4,5, Shujuan Guo 1,2,3, Junhou Hui 6, Peng Liu 6, Litao Yang 4, Sheng-Ce Tao 1,2,3
1Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 2State Key Laboratory of Oncogenes and Related Genes, 3School of Biomedical Engineering, Shanghai Jiao Tong University, 4Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 5Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, 6Department of Biomedical Engineering, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

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Neuroscience

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons
Mengye Zhu 1, Daying Zhang 1, Sicong Peng 2, Nana Liu 2, Jing Wu 2, Haixia Kuang 2, Tao Liu 2,3
1Department of Pain, First Affiliated Hospital of Nanchang University, 2Department of Pediatrics, First Affiliated Hospital of Nanchang University, 3Center for Laboratory Medicine, First Affiliated Hospital of Nanchang University

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

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Medicine

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis
Kaiyue Zhang 1, Chen Wang 1, Ran Wang 2, Shang Chen 1, Zongjin Li 1
1Nankai University School of Medicine, 2State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

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Medicine

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice
Fuliang Luo 1, Xin Wang 1, Xiaokang Luo 1, Bo Li 1, Desheng Zhu 2, Hansong Sun 3, Yue Tang 1
1Beijing Key Laboratory of Pre-clinical Research and Evaluation for Cardiovascular Implant Materials, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 2Laboratory Animal Center, Peking University, 3Center of Cardiac Surgery for Adults, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

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Medicine

Technical Modification of the Terminal Ureter During Total Transperitoneal Laparoscopic Nephroureterectomy for Upper Urinary Tract Urothelial Carcinoma
Chunlong Fu *1,2, Yaqun Zhang *1, Xin Wang 1, Shengcai Zhu 1, Huimin Hou 1, Shengjie Liu 1, Cheng Pang 1, Xinda Song 1, Jia Chen 1, Jianye Wang 1, Ming Liu 1,2
1(Urology) Beijing Hospital, National Center of Gerontology, The Joint Unit of the Chinese Academy of Medical Sciences, 2Graduate School of Peking Union Medical College, Beijing Hospital

Here, we present a protocol to increase the surgical field of view and reduce the difficulty of total transperitoneal laparoscopic nephroureterectomy surgery by precutting the umbilical ligament before treating the terminal ureter.

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Medicine

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases
Xiaofan Jia *1,2, Ling He *1,3, Yong Gu 1, Hilary High 1, Liping Yu 1
1Barbara Davis Center for Diabetes, University of Colorado School of Medicine, 2Department of Endocrinology, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, 3Department of endocrinology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

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Medicine

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery
Tao Liu *1,2,3,4,5, Xueling Song *1,2,3,4,5, Xiaoying Zheng *1,2,3,4,5, Liying Yan 1,2,3,4,5, Caihong Ma 1,2,3,5, Rong Li 1,2,3,4,5, Jie Yan 1,2,3,4,5, Jie Qiao 1,2,3,4,5
1Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, 2National Clinical Research Center for Obstetrics and Gynecology, 3Key Laboratory of Assisted Reproduction, Ministry of Education, 4Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, 5Research Units of Comprehensive Diagnosis and Treatment of Oocyte Maturation Arrest, Chinese Academy of Medical Sciences

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

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Medicine

Combined Endoscopic and Transoral Approach for Total Maxillectomy
Lun Xu 1, Ke-qing Zhao 1, Hong-meng Yu 1,2
1Department of Otolaryngology, Eye & ENT Hospital, Fudan University, 2Research Units of New Technologies of Endoscopic Surgery in Skull Base Tumor, Chinese Academy of Medical Sciences

Combined Endoscopic and Transoral Approach for Total Maxillectomy

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Biology

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium
Meng Zhang 1,2, Mohamed N. Seleem 3, Ji-Xin Cheng 1,2,4,5
1Department of Electrical and Computer Engineering, Boston University, 2Boston University Photonics Center, Boston University, 3Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 4Department of Biomedical Engineering, Boston University, 5Department of Chemistry, Boston University

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

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Neuroscience

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells
Sue Han *1, Xin Wang *1, Nicholas Cordero 1, Ling-Gang Wu 1
1National Institute of Neurological Disorders and Stroke

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

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Immunology and Infection

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19
Xiaofan Jia *1, Ling He *1,2, Dongmei Miao 1, Caiguo Zhang 1, Marian Rewers 1, Liping Yu 1
1Barbara Davis Center for Diabetes, University of Colorado School of Medicine, 2Department of Endocrinology, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

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Immunology and Infection

Construction of Adenoviral Vectors using DNA Assembly Technology
Xiaohui Zou *1, Yun Zhu *2, Chenglong Li 1,3, Yali Duan 2, Linlin Zhang 2, Xiaojuan Guo 1, Wenzhe Hou 1, Zhengde Xie 2,4, Zhuozhuang Lu 1,5
1NHC Key Laboratory of Medical Virology and Viral Diseases, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 2Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health, 3Henan Chemical Technician College, 4Research Unit of Critical Infection in Childre, Chinese Academy of Medical Sciences, 5Chinese Academy of Sciences Joint Research Center for Emerging Infectious Diseases and Biosafety, Chinese Center for Disease Control and Prevention-Wuhan Institute of Virology

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

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Biology

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization
Dingyun Song 1,2, Yicong Chen 1,3, Xin Wang 1,4, Xueying Chen 1,2, Shuwei Gao 1,3, Wenxiu Xu 1,3, Shengnan Yang 5, Zai Wang 6, Liang Peng 7, Huaping Dai 1,2
1Department of Pulmonary and Critical Care Medicine, Center of Respiratory Medicine, China-Japan Friendship Hospital, National Clinical Research Center for Respiratory Diseases, Institute of Respiratory Medicine, Chinese Academy of Medical Sciences, 2Graduate School of Peking Union Medical College, Chinese Academy of Medical Science and Peking Union Medical College, 3Capital Medical University, 4Beijing University of Chinese Medicine, 5Tianjin Chest Hospital, 6Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, 7Beijing Key Laboratory for Immune-Mediated Inflammatory Diseases, Institute of Clinical Medical Sciences, China-Japan Friendship Hospital

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

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Immunology and Infection

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis
Linlin Zhang 1,2, Mengjia Liu 1,2, Meng Zhang 1,2, Junhong Ai 1,2, Jiao Tian 1,2, Ran Wang 1,2, Zhengde Xie 1,2
1Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, 2Research Unit of Critical Infection in Children, 2019RU016, Chinese Academy of Medical Sciences

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

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Immunology and Infection

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique
Linlin Zhang *1,2, Meng Zhang *1,2,3, Mengjia Liu 1,2, Junhong Ai 1,2, Jiao Tian 1,2, Huizheng Ge 4, Ran Wang 1,2, Zhengde Xie 1,2
1Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, 2Research Unit of Critical Infection in Children, 2019RU016, Chinese Academy of Medical Sciences, 3Department of Pediatric Rehabilitation, Beijing Boai Hospital, School of Rehabilitation Medicine, Capital Medical University, China Rehabilitation Research Center, 4Center of Excellence (Beijing), Becton Dickinson Medical Devices (Shanghai) Co Ltd

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (TPF) profiles in peripheral blood mononuclear cells (PBMCs) within Japanese encephalitis virus (JEV)-vaccinated children. The detection method and flow cytometry color scheme of JEV-specific TPFs were tested to provide a reference for similar studies.

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Immunology and Infection

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children
Huizheng Ge *1, Linlin Zhang *2,3, Mengjia Liu *2,3, Xi Yang 1, Xu Wu 1, Ran Wang 2,3, Zhengde Xie 2,3
1Center of Excellence (Beijing), Becton Dickinson Medical Devices (Shanghai) Co Ltd, 2Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, 3Research Unit of Critical Infection in Children, 2019RU016, Chinese Academy of Medical Sciences

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

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Medicine

Establishment of a Minimally Invasive Rat Model of Pulmonary Embolism Using Autologous Blood Clots
Ruihao Li 1,2, Zuoguan Chen 1, Yaming Guo 1,2, Changduo Wang 3, Yongpeng Diao 1, Jing Yang 3, Yongjun Li 1,2
1Department of Vascular Surgery, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, 2Graduate School of Chinese Academy of Medical Sciences & Peking Union Medical College, 3Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College

A detailed methodology for establishing a minimally invasive rat model of pulmonary embolism using autologous blood clots is described. Additional methods for quantifying the infarcted area and visualizing the pulmonary arterial tree are also provided.

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