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Shanghai Jiao Tong University

54 ARTICLES PUBLISHED IN JoVE

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Biology

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice
Yu Chen 1,2, Tambudzai Shamu 2, Hui Chen 3, Peter Besmer 3, Charles L. Sawyers 2,4, Ping Chi 1,5
1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

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Medicine

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging
Xiaobing Liu *1,2, Toya Terry *1, Su Pan 1, Zhongwei Yang 1, James T. Willerson 1, Richard A. F. Dixon 1, Qi Liu 1
1The Texas Heart Institute at St. Luke's Episcopal Hospital, 2Department of Vascular Surgery, The Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

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Neuroscience

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)
Ying Wang 1, Nicholas Baker 1, Gro V. Amdam 1,2
1School of Life Sciences, Arizona State University , 2Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

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Neuroscience

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons
Xiaobei Zhao 1, Yue Zhou 2, April M. Weissmiller 1, Matthew L. Pearn 3,4, William C. Mobley 1, Chengbiao Wu 1
1Department of Neurosciences, University of California, San Diego, 2School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, 3Department of Anesthesiology, University of California, San Diego, 4VA San Diego Healthcare System

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

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Medicine

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease
Bruce M. Damon 1,2,3,4, Ke Li 1,2, Richard D. Dortch 1,2, E. Brian Welch 1,2, Jane H. Park 1,2,4, Amanda K. W. Buck 1,2, Theodore F. Towse 1,2,5, Mark D. Does 1,2,3, Daniel F. Gochberg 1,2,6, Nathan D. Bryant 1,2
1Institute of Imaging Science, Vanderbilt University, 2Department of Radiology and Radiological Sciences, Vanderbilt University, 3Department of Biomedical Engineering, Vanderbilt University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Department of Physical Medicine and Rehabilitation, Vanderbilt University, 6Department of Physics and Astronomy, Vanderbilt University

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

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Neuroscience

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons
Melanie Martin 1,2, Ke Li 1, Megan C. Wright 2, Angelo C. Lepore 1
1Department of Neuroscience, Farber Institute for Neurosciences, Sidney Kimmel Medical College at Thomas Jefferson University, 2Department of Biology, Arcadia University

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

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Medicine

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging
Lei Li *1, Xiaoyun Guo *1, Yi Chen 1, Haifeng Yin 1, Jing Li 1, Jessica Doan 1, Qinghang Liu 1
1Department of Physiology and Biophysics, University of Washington

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

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Immunology and Infection

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging
Yucheng Wang 1,2,3, Olivia D. Harrington 1, Ying Wang 1, Clinton K. Murray 4, Michael R. Hamblin 1, Tianhong Dai 1
1Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, 2Department of Medical Oncology, Beijing Institute of Translational Medicine, Chinese Academy of Sciences, 3Cancer Center, Aviation General Hospital, Beijing, 4Infectious Disease Service, Brooke Army Medical Center

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

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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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Bioengineering

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation
Jing Zhu 1, Xiaowei Dong 1
1Department of Pharmaceutical Sciences, College of Pharmacy, University of North Texas Health Science Center

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

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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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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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Biochemistry

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay
Bo Han 1, Man Zhang 2, Peng Sun 1, Shangwei Hou 3
1Department of General Surgery, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, 2Key Laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University, 3Hongqiao International Institute of Medicine, Tongren Hospital, Shanghai Jiao Tong University School of Medicine

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

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Biology

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice
Yanan Fu *1,2, Wenlong Jiang *2, Yichao Zhao *2, Yuli Huang 1, Heng Zhang 1, Hongju Wang 1, Jun Pu 1,2
1Department of Cardiology, The First Affiliated Hospital of Bengbu Medical College, 2Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

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Cancer Research

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay
Ying Wang 1, DanQi Chen 1, MingYu Chen 1, KaiYuan Ji 1, DeXuan Ma 1, LiangFu Zhou 1
1Department of Neurosurgery, Huashan Hospital, Fudan University

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

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Cancer Research

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation
Juan Wang 1, Jin Wen 2, Xiao-Xiang Chen 1, Guang-Liang Chen 3
1Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 2Department of Nephrology and Rheumatology, Yongchuan Hospital of Chongqing Medical University, 3Department of Medical Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

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Genetics

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice
Jing-Ying Guo 1, Lu He 1, Teng-Fei Qu 1, Yu-Ying Liu 2, Ke Liu 1, Guo-Peng Wang 1, Shu-Sheng Gong 1
1Department of Otolaryngology-Head and Neck Surgery, Beijing Friendship Hospital, Capital Medical University, 2Department of Otolaryngology-Head and Neck Surgery, Shanghai First People's Hospital, Shanghai Jiao Tong University

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

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Cancer Research

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer
Larry C. Cheng *1,2, Zhen Li *3, Thomas G. Graeber 4, Nicholas A. Graham 5, Justin M. Drake 1,2,3,6,7
1Graduate Program in Cellular and Molecular Pharmacology, School of Graduate Studies, Rutgers University, The State University of New Jersey, 2Graduate Program in Quantitative Biomedicine, School of Graduate Studies, Rutgers University, The State University of New Jersey, 3Department of Medicine, Division of Medical Oncology, Rutgers Robert Wood Johnson Medical School, 4Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, UCLA Metabolomics Center, and California NanoSystems Institute, David Geffen School of Medicine, University of California, Los Angeles, 5Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, 6Pharmacology, Rutgers Robert Wood Johnson Medical School, 7Cancer Metabolism and Growth Program, Rutgers Cancer Institute of New Jersey

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

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Biochemistry

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation
Anne M. Scott 1, Ke Li 1, Weiming Li 1
1Department of Fisheries and Wildlife, Michigan State University

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

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Engineering

Radio Frequency Magnetron Sputtering of GdBa2Cu3O7δ/ La0.67Sr0.33MnO3 Quasi-bilayer Films on SrTiO3 (STO) Single-crystal Substrates
Ying Wang 1,2, Zhen Li 1,2, YongSheng Liu 1, Yijie Li 2, Linfei Liu 2, Da Xu 2, Xiaojing Luo 1, Tian Gao 1,3, Yanyan Zhu 1,4, Luozeng Zhou 3, Jianming Xu 3
1Department of Physics, Mathematics, Shanghai Key Laboratory of Materials Protection and Advanced Materials in Electric Power, Shanghai University of Electric Power, 2Key Laboratory of Artificial Structure and Quantum Control, Ministry of Education, Department of Physics, Shanghai Jiao Tong University, 3Shanghai Institute of Space Power-sources, 4Shanghai Key Laboratory of High Temperature Superconductors, Shanghai University

Here, we present a protocol to grow LSMO nanoparticles and (Gd) BCO films on (001) SrTiO3 (STO) single-crystal substrates by radio frequency (RF)-sputtering.

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Engineering

Fabrication of Soft Pneumatic Network Actuators with Oblique Chambers
Lisen Ge *1,2, Tianyu Wang *1,3, Ningbin Zhang 1,2, Guoying Gu 1,2
1State Key Laboratory of Mechanical System and Vibration, Shanghai Jiao Tong University, 2Robotics Institute, School of Mechanical Engineering, Shanghai Jiao Tong University, 3University of Michigan-Shanghai Jiao Tong University Joint Institute, Shanghai Jiao Tong University

Here we present a fabrication method of soft pneumatic network actuators with oblique chambers. The actuators are capable of generating coupled bending and twisting motions, which broadens their application in soft robotics.

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Immunology and Infection

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay
Caiyun Liu 1, Mingqin Zhu 1, Ying Wang 1
1Department of Neurology and Neuroscience Center, The First Hospital of Jilin University

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

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Engineering

Evaluating Targeting Accuracy in the Focal Plane for an Ultrasound-guided High-intensity Focused Ultrasound Phased-array System
Ke Li 1,2, Jingfeng Bai 1,2, Yazhu Chen 1,2, Xiang Ji 1,2
1Biomedical Instrument Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 2Shanghai Med-X Engineering Center for Medical Equipment and Technology, Shanghai Jiao Tong University

This study describes a protocol to evaluate the targeting accuracy in the focal plane of an ultrasound-guided high-intensity focused ultrasound phased-array system.

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Genetics

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy
Baoheng Gui *1,2,3, Yingxin Zhang *4, Bo Liang 5, Yvonne Ka Yin Kwok 4, Wai Ting Lui 4, Queenie Sum Yee Yeung 4, Lingyin Kong 6, Liming Xuan 6, Jacqueline Pui Wah Chung 4, Kwong Wai Choy 3,4
1Department of Genetics and Metabolism, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, 2Birth Defects Prevention and Control Institute of Guangxi Zhuang Autonomous Region, 3Shenzhen Research Institute, The Chinese University of Hong Kong, 4Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, 5State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 6Basecare Medical Device Co., Ltd

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

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Biology

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly
Chang Li *1,2, Zhu Hui Liu *1,2, Jia Wei Chen 1,2, Xin Yi Shu 1,2, Ying Shen 1, Feng Hua Ding 1, Rui Yan Zhang 1, Wei Feng Shen 1, Lin Lu 1, Xiao Qun Wang 1
1Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 2Institute of Cardiovascular Diseases, Shanghai Jiao Tong University

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

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Biology

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells
Shaoyang Wang 1,2, Minglie Hu 2, Tao Ren 1, Hao He 3
1Department of Respiratory Medicine, Shanghai Sixth People's Hospital, School of Medicine, Shanghai Jiao Tong University, 2Ultrafast Laser Laboratory, College of Precision Instrument and Optoelectronics Engineering, Tianjin University, 3School of Biomedical Engineering, Shanghai Jiao Tong University

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

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Genetics

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants
Ying Wang *1,2, Zheng Kuang *1,2, Lei Li 2, Xiaozeng Yang 1
1Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, 2State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Advanced Agricultural Sciences and School of Life Sciences, Peking University

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

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Immunology and Infection

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models
Soichi Sano 1, Ying Wang 1, Megan A. Evans 1, Yoshimitsu Yura 1, Miho Sano 1, Hayato Ogawa 1, Keita Horitani 1, Heather Doviak 1, Kenneth Walsh 1
1Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

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Neuroscience

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System
Yi Chen *1, Curt Mazur *2, Yi Luo *1, Linhong Sun 1, Mingdi Zhang 1, Alex McCampbell 1, Giulio Srubek Tomassy 1
1Biogen, Inc., 2Ionis Pharmaceuticals

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

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Developmental Biology

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue
Yiding Qi *1, Xiang Miao *2, Lian Xu 3, Mengxia Fu 3, Shi Peng 4, Kailei Shi 5, Jun Li 4, Maoqing Ye 5, Ruogu Li 1
1Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiao Tong University, 2Shanghai Institute of Nutrition and Health, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 3Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 4Department of Cardiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 5Department of Cardiology, Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital Affiliated to Fudan University

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

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JoVE Core

Fabrication of Compressed Hosiery and Measurement of its Pressure Characteristic Exerted on the Lower Limbs
Guangwu Sun 1,2,3, Jiecong Li 4, Xiaona Chen 1,2,3, Yanmei Li 1,2,3, Yu Chen 1, Qi Fang 1, Hong Xie 1,2,3
1School of Fashion Engineering, Shanghai University of Engineering Science, 2Textile Industrial Key Laboratory of Ergonomics and Functional Clothing, Shanghai University of Engineering Science, 3Sino-British Joint Lab for Smart Sportswear, Shanghai University of Engineering Science, 4College of Textiles, Donghua University

This article reports fabrication, structure and pressure measurement of compressed hosiery by employing direct and indirect methods.

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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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Biology

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice
Dawei Xu *1,2, Palash Chandra Mondol *1, Muhammad Uzair 1, Matthew R. Tucker 3, Dabing Zhang 1,3
1Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University-University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 2College of Agronomy, Anhui Agricultural University, Heifei, China, 3School of Agriculture, Food and Wine, University of Adelaide

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

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JoVE Core

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis
Eunbee Park 1, Megan A. Evans 4, Heather Doviak 4, Keita Horitani 4, Hayato Ogawa 4, Yoshimitsu Yura 4, Ying Wang 2, Soichi Sano 3,4, Kenneth Walsh 1,4
1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 2Department of Cardiology, Xinqiao Hospital, Army Medical University, 3Department of Cardiology, Osaka City University Graduate School of Medicine, 4Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

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Biochemistry

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus
Yunrong Gao 1, Claire Ogilvie 1, Anirudh Raghavan 1, Chloe Von Hoffmann 1, Bo Liang 1
1Department of Biochemistry, Emory University School of Medicine

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

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Medicine

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.
Babak Saravi *1,2, Gernot Lang *2, Sibylle Grad 1, Mauro Alini 1, R. Geoff Richards 1,2, Hagen Schmal 2, Norbert Südkamp 2, Zhen Li 1
1AO Research Institute Davos, Davos, Switzerland, 2Department of Orthopedics and Trauma Surgery, Medical Centre-Albert-Ludwigs-University of Freiburg, Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Freiburg, Germany

This protocol presents a novel experimental model of proinflammatory, degenerative bovine organ culture to simulate early-stage intervertebral disc degeneration.

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Cancer Research

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice
Lisha Wang 1, Zhiming Wang 1, Junyi Guo 2, Huayu Lin 1, Shuqiong Wen 2, Qiao Liu 1, Yiding Li 3, Qing Wu 1, Leiqiong Gao 1, Xiangyu Chen 1, Luoyingzi Xie 1, Qin Tian 1, Jianfang Tang 1, Zhirong Li 1, Li Hu 1, Juan Wang 4, Lifan Xu 1, Qizhao Huang 5, Lilin Ye 1
1Institute of Immunology, Third Military Medical University, 2Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Stomatological Hospital, Sun Yat-Sen University, 3Shigatse Branch, Xinqiao Hospital, Third Military Medical University, 4Department of Emergency Medicine, Southwest Hospital, Third Military Medical University, 5Cancer Center, The General Hospital of Western Theater Command

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

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Medicine

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee
Xiang Ji 1, Akihiro Nakahata 2, Zixi Zhao 2, Hiroshi Kuroki 2, Tomoki Aoyama 1, Akira Ito 2
1Department of Development and Rehabilitation of Motor Function, Human Health Sciences, Graduate School of Medicine, Kyoto University, 2Department of Motor Function Analysis, Human Health Sciences, Graduate School of Medicine, Kyoto University

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

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Medicine

A Modified Simple Method for Induction of Myocardial Infarction in Mice
Chenglin Jiang *1,2, Jianqing Chen *1,2, Yichao Zhao *2, Dasheng Gao 1, Hongju Wang 1, Jun Pu 1,2
1Department of Cardiology, The First Affiliated Hospital of Bengbu Medical College, 2Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

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Neuroscience

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging
Rashmi Thapa 1, Bo Liang 2, Rongsong Liu 3, Yun Li 1
1Department of Zoology and Physiology, University of Wyoming, 2School of Electrical Engineering & Computer Science, University of North Dakota, 3Department of Mathematics and Statistics, University of Wyoming

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

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Biology

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion
Chao E *1, Liqiang Dai *1,2, Jiaqi Tian 3,4, Lin-Tai Da 4, Jin Yu 5,6,7
1Beijing Computational Science Research Center, 2Shenzhen JL Computational Science and Applied Research Institute, 3School of Medical Informatics and Engineering, Xuzhou Medical University, 4Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 5Department of Physics and Astronomy, University of California, Irvine, 6Department of Chemistry, University of California, Irvine, 7NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine

The goal of this protocol is to reveal structural dynamics of one-dimensional diffusion of protein along DNA, using a plant transcription factor WRKY domain protein as an exemplary system. To do this, both atomistic and coarse-grained molecular dynamics simulations along with extensive computational samplings have been implemented.

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Bioengineering

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography
Yuan Feng 1, Suhao Qiu 1, Yu Chen 1, Runke Wang 1, Zhao He 1, Linghan Kong 1, Yi Chen 1, Shengyuan Ma 1
1School of Biomedical Engineering, Shanghai Jiao Tong University

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

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Biochemistry

Visualizing Yeast Organelles with Fluorescent Protein Markers
Chang-Yue Liu 1, Jing Zhu 1, Zhiping Xie 1
1State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

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Bioengineering

Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology
Sheng-Chang Luo 1,2, Ying Wang 3, Ranjith Kumar Kankala 1,2, Yu Shrike Zhang 4, Ai-Zheng Chen 1,2
1Institute of Biomaterials and Tissue Engineering, Huaqiao University, 2Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, 3Affiliated Dongguan Hospital, Southern Medical University, 4Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

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Biology

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans
Marzia Savini *1,2, Yi-Tang Lee 2,3,4, Meng C. Wang 2,4,5, Yue Zhou *2
1Graduate Program in Developmental Biology, Baylor College of Medicine, 2Huffington Center on Aging, Baylor College of Medicine, 3Integrative Program of Molecular and Biochemical Sciences, Baylor College of Medicine, 4Department of Molecular and Human Genetics, Baylor College of Medicine, 5Howard Hughes Medical Institute, Baylor College of Medicine

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

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Neuroscience

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala
Yongchang Lu *1,2, Yang Dai *1,2, Siqi Ou 1,2, Yujing Miao 1, Ying Wang 1,2, Quanlei Liu 1,2, Yihe Wang 1,2, Penghu Wei 1,2, Yongzhi Shan 1,2, Guoguang Zhao 1,2,3
1Department of Neurosurgery, Xuanwu Hospital Capital Medical University, 2China Clinical Research Center for Epilepsy Capital Medical University, 3China Beijing Municipal Geriatric Medical Research Center

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

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Biology

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay
Ying Wang 1,2, Zhuo An 1,2, Zhifang Zhao 1,2, Can Li 3, Jiangbo Fan 1,2
1Shanghai Collaborative Innovation Center of Agri-Seeds, School of Agriculture and Biology, Shanghai Jiao Tong University, 2Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, 3Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

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Biology

Isolation and Transcriptome Analysis of Plant Cell Types
Jun Zhang 1, Mayra Ahmad 1, Rongrong Xie 1, Hongbo Gao 1
1Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

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Medicine

Establishing a Device for Sleep Deprivation in Mice
Jianqing Chen *1, Jingze Wei *1, Xiaoying Ying *2, Fan Yang *2, Yichao Zhao *2, Jun Pu 1,2
1Department of Cardiology, The First Affiliated Hospital of Bengbu Medical College, 2Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University

The present protocol outlines a method for setting up a cost-effective rocker platform-based device used for inducing sleep deprivation in mice. This device has proven to be effective in causing disruptions in electroencephalogram (EEG)-evidenced sleep patterns, as well as inducing metabolic and molecular changes associated with sleep deprivation.

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Medicine

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model
Sijuan Sun *1, Hongbin Zhu *2, Shoubao Wang 3, Xiuxia Xu 4, Lincai Ye 2,5,6
1Department of Pediatric Intensive Care Unit, Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiaotong University, 2Department of Thoracic and Cardiovascular Surgery, Shanghai Children’s Hospital, School of Medicine, Shanghai Jiaotong University, 3Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, 4Department of Radiology, Huangpu Branch, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 5Institute of Pediatric Translational Medicine, Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiaotong University, 6Shanghai Institute for Pediatric Congenital Heart Disease, Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiaotong University

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.

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Medicine

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement
Yuanqi Liu *1,2,3,4,5, Siyuan Sun *1,2,3,4,5, Ziyi Jiang *6, Xinyi Gong 1,2,3,4,5, Yiling Yang 1,2,3,4,5, Yanfei Zhu 1,2,3,4,5, Hongyuan Xu 1,2,3,4,5, Anting Jin 1,2,3,4,5, Xiangru Huang 1,2,3,4,5, Xin Gao 1,2,3,4,5, Tingwei Lu 1,2,3,4,5, Jingyi Liu 1,2,3,4,5, Xinyu Wang 1,2,3,4,5, Qinggang Dai 2,3,4,5,7, Lingyong Jiang 1,2,3,4,5
1Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 2College of Stomatology, Shanghai Jiao Tong University, 3National Center for Stomatology, 4National Clinical Research Center for Oral Diseases, 5Shanghai Key Laboratory of Stomatology, 6Shanghai Starriver Bilingual School, 7The 2nd Dental Center, Ninth People’s Hospital, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

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Biology

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model
Ziduan Ding 1,2, Ruomei Li 1,2, Yufeng Duan 1,2, Zhenxia Li 1,2, Bing Fang 1,2, Dian Jing 1,2
1Department of Orthodontics, China Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, 2National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai Jiao Tong University

This protocol presents a standardized suture expansion mouse model and a 3-D visualization method to study the mechanobiological changes of the suture and bone remodeling under tensile force loading.

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Developmental Biology

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection
Jianan Tang 1,2,3, Baoli Zhang 1,2,3, Chunhua Tang 4,5,6, Xuemei Wang 1,2,3, Minmin Hua 1,2,3, Yuchuan Zhou 4,5,6, Huijuan Shi 1,2,3, Tiancheng Zhang 1,2,3
1Shanghai-MOST Key Laboratory of Health and Disease Genomics, 2NHC Key Lab of Reproduction Regulation, 3Shanghai Institute for Biomedical and Pharmaceutical Technologies, 4International Peace Maternity and Child Health Hospital, 5School of Medicine, Shanghai Jiao Tong University, 6Shanghai Key Laboratory of Embryo Original Diseases

Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

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Bioengineering

Motor Imagery Performance Through Embodied Digital Twins in a Virtual Reality-Enabled Brain-Computer Interface Environment
Kishor Lakshminarayanan 1, Rakshit Shah 2, Vadivelan Ramu 1, Deepa Madathil 3, Yifei Yao 4, Inga Wang 5, Brahim Brahmi 6, Mohammad Habibur Rahman 7
1Department of Sensors and Biomedical Tech, School of Electronics Engineering, Vellore Institute of Technology, 2Department of Orthopaedic Surgery, University of Arizona, 3Jindal Institute of Behavioural Sciences, O. P. Jindal Global University, 4Soft Tissue Biomechanics Laboratory, Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 5Department of Occupational Science & Technology, University of Wisconsin-Milwaukee, 6Electrical Engineering, Collège Ahuntsic, 7Department of Mechanical Engineering, BioRobotics Lab, University of Wisconsin-Milwaukee

Motor imagery in a virtual reality environment has wide applications in brain-computer interface systems. This manuscript outlines the use of personalized digital avatars that resemble the participants performing movements imagined by the participant in a virtual reality environment to enhance immersion and a sense of body ownership.

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