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Peking University

51 ARTICLES PUBLISHED IN JoVE

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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

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons
Ji-Yong Kang 1, Daichi Kawaguchi 2, Lei Wang 3
1Department of Neuroscience, School of Medicine, Tufts University, 2Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 3Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

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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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Cancer Research

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases
Mercedes Rodriguez-Teja 1, Claudia Breit 2, Mitchell Clarke 3, Kamil Talar 3, Kai Wang 3, Mohammad A. Mohammad 3, Sage Pickwell 3, Guillermina Etchandy 1, Graeme J. Stasiuk 3, Justin Sturge 3
1Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), 2Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 3School of Biological, Biomedical & Environmental Sciences, University of Hull

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

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Bioengineering

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior
Kai Wang 1, Kam W. Leong 2, Yong Yang 1
1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Biomedical Engineering, Columbia University

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

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Bioengineering

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice
Kai Wang *1, Xi Wang *1, Cheng-sheng Han *2, Liang-yi Chen 2, Ying Luo 1
1Department of Biomedical Engineering, Peking University, 2Institute of Molecular Medicine, Peking University

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

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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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Biology

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells
Ke Zou 1,2, Diana S. Ren 2, Qi Ou-yang 1,3, Hao Li 2, Jiashun Zheng 2
1The State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics, Peking University, 2Department of Biochemistry and Biophysics, University of California, San Francisco, 3Peking-Tsinghua Center for Life Sciences at Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

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Environment

Data Processing Methods for 3D Seismic Imaging of Subsurface Volcanoes: Applications to the Tarim Flood Basalt
Lei Wang 1, Wei Tian 2, Yongmin Shi 2
1College of Engineering, Peking University, 2School of Earth and Space Sciences, Peking University

Three-dimensional (3D) reflection seismology is a powerful method for imaging subsurface volcanoes. By using industrial 3D seismological data from the Tarim Basin, we illustrate how to extract the sills and the conduits of subsurface volcanoes from seismic data cubes.

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Developmental Biology

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation
Zhe Liu 1,2, Siling Fu 2,3, Nan Tang 2
1Department of Life Sciences, Peking University, 2National Institute of Biological Sciences, Beijing, 3Graduate School of Peking Union Medical College

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

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Neuroscience

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters
Long Mei 1,2, Cheng Zhan 2, Eric Erquan Zhang 2
1PTN Joint Graduate Program, School of Life Sciences, Peking University, 2National Institute of Biological Sciences, Beijing

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

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Behavior

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor
Xing Chen 1, Detlef Wolf 1, Juliane Siebourg-Polster 2, Christian Czech 3, Ulrike Bonati 4,5, Dirk Fischer 4,5, Omar Khwaja 6, Martin Strahm 1
1Data Science, Roche Pharmaceutical Research and Early Development Informatics, Roche Innovation Center Basel, F. Hoffmann-La Roche, Ltd., 2Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Ltd., 3Biomarker Experimental Medicine, Neuroscience, Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Ltd., 4Division of Neuropediatrics, University of Basel Children's Hospital, 5Department of Neurology, University of Basel Hospital, 6Translational Medicine, Neuroscience and Rare Diseases, Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche, Ltd.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

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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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JoVE Journal

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy
Jing Guo 1, Sifan You 1, Zhichang Wang 1, Jinbo Peng 1, Runze Ma 1, Ying Jiang 1,2
1International Center for Quantum Materials, School of Physics, Peking University, 2Collaborative Innovation Center of Quantum Matter

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

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Biology

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model
Kang Qin 1, Yifan Wu 1, Yiwa Pan 1, Kai Wang 1, Deling Kong 1, Qiang Zhao 1
1State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials (Ministry of Education), College of Life Sciences, Nankai University

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

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Cancer Research

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry
Xiaoyu An *1,2, Xuesong Ouyang *1, Hui Zhang 1, Tingting Li 1, Yu-yang Huang 1, Zhiyuan Li 1, Demin Zhou 2, Qi-Xang Li 1,2
1Crown Bioscience Inc., 2State Key Laboratory of Natural and Biomimetic Drugs, Peking University

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200–600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

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Developmental Biology

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells
Lin-Chen Li *1,2, Xin-Xin Yu *1,2, Yu-Wei Zhang *1, Ye Feng *1,3, Wei-Lin Qiu *1,3, Cheng-Ran Xu 1
1College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, 2Academy for Advanced Interdisciplinary Studies, Peking University, 3PKU-Tsinghua-NIBS Graduate Program, Peking University

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

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Developmental Biology

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart
Chenglu Xiao 1, Fang Wang 2, Junjie Hou 1, Xiaojun Zhu 1, Ying Luo 2, Jing-Wei Xiong 1
1Beijing Key Laboratory of Cardiometabolic Molecular Medicine, State Key Laboratory of Natural and Biomimetic Drugs, Institute of Molecular Medicine, Peking University, 2Department of Biomedical Engineering, College of Engineering, Peking University

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

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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

Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing
Matthews M. Springer 1, Benjamin D. Strycker 1,2, Kai Wang 1, Alexei V. Sokolov 1,2, Marlan O. Scully 1,2
1Texas A&M University, 2Baylor University

High-intensity femtosecond pulses of laser light can undergo cycles of Kerr self-focusing and plasma defocusing, propagating an intense sub-millimeter-diameter beam over long distances. We describe a technique for generating and using these filaments to perform remote imaging and sensing beyond the classical diffraction limits of linear optics.

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Biochemistry

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy
Rongbing Huang *1,2, Mengting Han *1,2, Liying Meng 1,3, Xing Chen 1,2,3,4,5
1College of Chemistry and Molecular Engineering, Peking University, 2Beijing National Laboratory for Molecular Sciences, Peking University, 3Peking-Tsinghua Center for Life Sciences, Peking University, 4Synthetic and Functional Biomolecules Center, Peking University, 5Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

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Behavior

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats
Lupeng Yue 1,2, Fengrui Zhang 3, Xuejing Lu 1,2, You Wan 4,5,6, Li Hu 1,2,7
1CAS Key Laboratory of Mental Health, Institute of Psychology, 2Department of Psychology, University of Chinese Academy of Sciences, 3Research Center of Brain Cognitive Neuroscience, Liaoning Normal University, 4Neuroscience Research Institute, Peking University, 5Department of Neurobiology, School of Basic Medical Sciences, Peking University, 6Key Laboratory for Neuroscience, Ministry of Education/National Health and Family Planning Commission, Peking University, 7Department of Pain Management, State Key Clinical Specialty in Pain Medicine, Second Affiliated Hospital of Guangzhou Medical University

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

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Neuroscience

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan
Qing-Ran Bai *1, Lu Dong *2, Qin Shen 1
1Brain and Spinal Cord Innovative Research Center of Tongji Hospital, School of Life Sciences and Technology, Tongji University and Frontier Science Research Center for Stem Cells of Ministry of Education, 2College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Sciences, Beijing National Laboratory for Molecular Sciences, Synthetic and Functional Biomolecules Center, and Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

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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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JoVE Journal

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure
Honghui Mao *1,2, Xiuchao Wang *1,3, Wen Chen 4, FengYu Liu 5, You Wan 5, Sanjue Hu 1, Junling Xing 1,6
1Department of Neurobiology, School of Basic Medicine, Fourth Military Medical University, 2Department of Toxicology, School of Public Health, ShanXi Medical University, 3Department of Psychology, Fourth Military Medical University, 4Department of Neurobiology, School of Basic Medical Sciences, Advanced Innovation Center for Human Brain Protection, Capital Medical University, 5Neuroscience Research Institute, Key Lab for Neuroscience, Ministry of Education/National Health Commission, Peking University, 6Department of Radiation Biology, Faculty of Preventive Medicine, Fourth Military Medical University

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system's relationship with pain.

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Biology

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana
Kai Wang 1, Kayin Law 1, Manhin Leung 1, Waishing Wong 1, Ning Li 1,2
1Division of Life Science, Energy Institute, Institute for the Environment, The Hong Kong University of Science and Technology, 2Shenzhen Research Institute, The Hong Kong University of Science and Technology

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

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Genetics

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses
Yuyang Wang *1, Weidi Xu *1,2, Huancheng Guo 1, Wenjie Gong 1, Biao He 1, Zhongzhong Tu 1, Changchun Tu 1,3, Ye Feng 1
1Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, 2Jilin Agricultural University, 3Jiangsu Co-innovation Centre for Prevention and Control of Important Animal Infectious Diseases and Zoonoses

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

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Cancer Research

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment
Lei Wang *1, Huan Chen *2,3, Fei Fei *1, Xianfeng He 2,3, Shaoyang Sun 1, Kunpeng Lv 1, Bo Yu 2,3, Jiang Long 2,4,5,6, Xu Wang 1
1Department of Biochemistry and Molecular Biology, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, School of Basic Medical Sciences, Fudan University, 2National Human Genetic Resources Sharing Service Platform (2005DKA21300), 3Fudan University Shanghai Cancer Center, 4Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, 5Department of Oncology, Shanghai Medical College, Fudan University, 6Pancreatic Cancer Institute, Fudan University

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

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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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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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Engineering

Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes
Xiang Zhang *1,2, Zhaolong Chen *3, Hongliang Chang 1,2, Jianchang Yan 1,2, Shenyuan Yang 2,4, Junxi Wang 1,2, Peng Gao 5, Tongbo Wei 1,2
1State Key Laboratory of Solid-State Lighting, Institute of Semiconductors, Chinese Academy of Sciences, 2Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Science, 3Center for Nanochemistry (CNC), Beijing National Laboratory for Molecular Science, College of Chemistry and Molecular Engineering, Peking University, 4State Key Laboratory of Superlattices and Microstructures, Institute of Semiconductors, Chinese Academy of Sciences, 5Electron Microscopy Laboratory, School of Physics, Peking University

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

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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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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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Cancer Research

Characterization and Functional Prediction of Bacteria in Ovarian Tissues
Lanbo Zhao *1, Weichu Zhao *2, Qi Wang *1, Dongxin Liang 1, Yu Liu 3, Guoxing Fu 4, Lu Han 1, Yiran Wang 1, Chao Sun 1, Qing Wang 1, Qing Song 1, Qiling Li 1, Qinrui Lu 1
1Department of Obstetrics and Gynecology, First Affiliated Hospital of Xi’an Jiaotong University, 2School of Social Sciences, University of Manchester, 3Department of Pathology, First Affiliated Hospital of Xi’an Jiaotong University, 4Omega Bioservices Inc

Immunohistochemistry staining and 16S ribosomal RNA gene (16S rRNA gene) sequencing were performed in order to discover and distinguish bacteria in cancerous and noncancerous ovarian tissues in situ. The compositional and functional differences of the bacteria were predicted by using BugBase and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt).

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Biology

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells
Boheng Liu 1,2, Nadine Mundt 3,4, Melissa Miller 1, David E. Clapham 5, Yuriy Kirichok 6, Polina V. Lishko 1
1Department of Molecular and Cell Biology, University of California, Berkeley, 2Department of Neurobiology, Peking University, 3Institute for Biology II / Chemosensation Lab, RWTH Aachen University, 4Research Training Group 2416 MultiSenses-MultiScales, RWTH Aachen University, 5Howard Hughes Medical Institute, Janelia Research Campus, 6Department of Physiology, University of California, San Francisco

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

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Genetics

A Virtual Machine Platform for Non-Computer Professionals for Using Deep Learning to Classify Biological Sequences of Metagenomic Data
Zhencheng Fang 1,2, Hongwei Zhou 1,3
1Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, 2Center for Quantitative Biology, Peking University, 3State Key Laboratory of Organ Failure Research, Southern Medical University

This tutorial describes a simple method to construct a deep learning algorithm for performing 2-class sequence classification of metagenomic data.

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Biochemistry

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA
Linyu Zuo *1, Jiawei Ding *1, Zhi Qi 1
1Center for Quantitative Biology, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

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Bioengineering

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632
Zhiwei Xie 1,2,3, Jizhou Shi 4, Min Zong 3, Qiuping Xu 1, Chang Liu 1, Jie Wen 1, Qun Zhang 1, Panpan Liu 1, Guanyi Liu 1, Jing Guo 2,5, Xunwei Wu 1
1Department of Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 2Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 3Department of Stomatology, Shengli Oilfield Central Hospital, 4Department of Pediatric Surgery, Shengli Oilfield Central Hospital, 5Ningbo Stomatology Hospital of Savaid Stomatology School, Hangzhou Medical College

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

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Neuroscience

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice
Lianteng Zhi 1, Jingyu Zhao 1, David Jaffe 1, Yuanxin Chen 1, Ninghan Wang 1,2, Qi Qin 1, Erin L. Seifert 3, Chenjian Li 4, Hui Zhang 1
1Department of Neuroscience, Thomas Jefferson University, 2School of Biomedical Engineering, Drexel University, 3Department of Pathology, MitoCare Center, Thomas Jefferson University, 4School of Life Sciences, Peking University

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.

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Medicine

Augmented Reality Navigation-Guided Core Decompression for Osteonecrosis of Femoral Head
Qi Wang *1,3, Qiuyuan Wang *2,3, Ran Ding 3, Youjie Yao 4, Junjun Pan 5, Weiguo Wang 3
1China-Japan Friendship School of Clinical Medicine, Peking University, 2Graduate School of Beijing University of Chinese Medicine, 3Department of Orthopedic Surgery, Beijing Key Lab of Immunes-Mediated Inflammatory Disease, China-Japan Friendship Hospital, 4Beijing Normal University, 5State Key Laboratory of Virtual Reality Technology and Systems, Beihang University

Augmented reality technology was applied to core decompression for osteonecrosis of the femoral head to realize real-time visualization of this surgical procedure. This method can effectively improve the safety and precision of core decompression.

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Medicine

A Modified Vessel-Sparing Microsurgical Vasoepididymostomy
Liang Zhou 1, Xingzhe Ji 1, Lei Wang 1
1Assisted Reproduction Center, Northwest Women’s and Children’s Hospital

Here, we present a protocol to preserve the vasal vessels in microsurgical vasoepididymostomy. The surgical security is enhanced by preserving the vasal vessels using a retrograde-anterograde dissociation and fixing the vasal vessels.

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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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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

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland
Xiangdi Mao 1, Sainan Min 2, Qihua He 3, Xin Cong 1
1Department of Physiology and Pathophysiology, Peking University School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, and Beijing Key Laboratory of Cardiovascular Receptors Research, 2Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Reseah Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, 3State Key Laboratory of Natural and Biomimetic Drugs, Peking University

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

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Biology

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model
Siting Zhang *1, Zeyu Cai *1, Xu Zhang 1, Tianfeng Ma 2, Wei Kong 1
1Department of Physiology and Pathophysiology, School of Basic Medical Sciences; Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Peking University, 2Department of Vascular and Endovascular Surgery, the First Medical Centre, Chinese PLA General Hospital

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

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Cancer Research

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model
Qi Wang 1, Chun-Hao Chen 1, Hongbo Xu 2, Sylvie Deborde 1,3, Richard J. Wong 1,3
1Department of Surgery, Memorial Sloan Kettering Cancer Center, 2Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, 3David M. Rubenstein Center for Pancreatic Cancer Research, Memorial Sloan Kettering Cancer Center

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

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Behavior

An Automated Method for Assessing Visual Acuity in Infants and Toddlers Using an Eye-Tracking System
Jing Wen 1, Bikun Yang 2, Jinshi Cui 2, Li Wang 3, Xiaoqing Li 1
1Department of Pediatric Ophthalmology, Peking University First Hospital, Peking University Children Vison Institute, Peking University, 2School of Intelligence Science and Technology, Peking University, 3School of Psychological and Cognitive Sciences and Beijing Key Laboratory of Behavior and Mental Health, Peking University

In this paper, we present a novel automated method for assessing visual acuity in infants and toddlers by using an eye-tracking system.

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Engineering

Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection
Guanzhou Lin 1, Jia Zhu 2, Yang Wang 1, Bingquan Yang 1,3, Shisong Xiong 1,3, Jinwen Zhang 1, Wengang Wu 1
1National Key Laboratory of Advanced Micro and Nano Manufacture Technology, School of Integrated Circuits, Peking University, 2School of Electronic Engineering and Computer Science, Peking University, 3School of Software and Microelectronics, Peking University

This protocol describes a fabrication method for a flexible substrate for surface-enhanced Raman scattering. This method has been used in the successful detection of low concentrations of R6G and Thiram.

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Biology

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation
Xiaochun Yang *1,2,3, Daichao Chen *4, Xin Dang *1,2,3, Jue Zhang 4,5, Yang Zhao 1,2,3,6
1State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 2MOE Key Laboratory of Cell Proliferation and Differentiation, Peking University, 3Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, College of Future Technology, Peking University, 4Academy for Advanced Interdisciplinary Studies, Peking University, 5College of Engineering, Peking University, 6Peking-Tsinghua Center for Life Sciences, Peking University

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

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Biochemistry

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining
Shuang Zhang *1, Yuqing Wang *2, Zheng Wang 1, Yihan Liu 2, Jingjing Cui 2, Yuxin Su 2, Jingyan Han 1, Jia Wang 2, Wanzhu Bai 2
1Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, 2Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor β after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

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