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The Medical College of Wisconsin

9 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

High-throughput Detection Method for Influenza Virus
Pawan Kumar 1, Allison E. Bartoszek 1, Thomas M. Moran 2, Jack Gorski 3, Sanjib Bhattacharyya 4, Jose F. Navidad 4, Monica S. Thakar 1,5, Subramaniam Malarkannan 1,6
1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine , 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin , 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens.

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Biology

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction
Li Wang 1, Graeme K. Carnegie 1
1Department of Pharmacology, University of Illinois at Chicago

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

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Biology

Massively Parallel Reporter Assays in Cultured Mammalian Cells
Alexandre Melnikov 1, Xiaolan Zhang 1, Peter Rogov 1, Li Wang 1, Tarjei S. Mikkelsen 1
1Broad Institute

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

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Developmental Biology

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5
Li Wang 1, Ziqing Liu 1, Chaoying Yin 1, Yang Zhou 1, Jiandong Liu 1, Li Qian 1
1Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

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Immunology and Infection

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells
Arash Nanbakhsh 1, Brad Best 2, Matthew Riese 3, Sridhar Rao 4, Li Wang 5, Jeffrey Medin 6, Monica S. Thakar 6, Subramaniam Malarkannan 1,5,6,7
1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, The Blood Center of Wisconsin, 2Vector Core Lab, Blood Research Institute, The Blood Center of Wisconsin, 3Laboratory of Lymphocyte Biology, Blood Research Institute, The Blood Center of Wisconsin, 4Laboratory of Stem Cell Transcriptional Regulation, Blood Research Institute, The Blood Center of Wisconsin, 5Department of Microbiology and Immunology, The Medical College of Wisconsin, 6Department of Pediatrics, The Medical College of Wisconsin, 7Department of Medicine, The Medical College of Wisconsin

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

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Bioengineering

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution
Nan Chen 1,2, Kai Zhu 1,2, Shiqiang Yan 3,4, Junmin Li 1,2, Tianyi Pan 4, Mieradilijiang Abudupataer 1,2, Fazle Alam 4, Xiaoning Sun 1,2, Li Wang 3,4, Chunsheng Wang 1,2
1Department of Cardiac Surgery and Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 2Shanghai Institute of Cardiovascular Diseases, 3Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, 4Institutes of Biomedical Sciences and Department of Systems Biology for Medicine, Shanghai Medical College, Fudan University

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

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Bioengineering

Microfluidic Model to Mimic Initial Event of Neovascularization
Ping Zhao 1, Xing Zhang 1, Xiao Liu 1, Li Wang 4, Haoran Su 1, Liyi Wang 1, Dongrui Zhang 1, Xiaoyan Deng 3, Yubo Fan 1,2
1Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Chinese Education Ministry, School of Biological Science and Medical Engineering, Beihang University, 2School of Engineering Medicine, Beihang University, 3Artificial Intelligence Key Laboratory of Sichuan Province, School of Automation and Information Engineering, Sichuan University of Science and Engineering, 4Beijing Research Center of Urban System Engineering

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

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Genetics

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform
Chengming Xu *1, Riqing Wei *2, Hui Lin 1, Leiyu Deng 1, Li Wang 3, Deyang Li 4, Honghui Den 5, Wensong Qin 1, Ping Wen 1, Ying Liu 1, Yingsong Wu 2, Qiang Ma 2, Jinliang Duan 1
1Centre for Women, Children, and Reproduction, Guangxi Key Laboratory of Metabolic Diseases Research, 2Department of Biopharmaceutics, School of Laboratory Medicine and Biotechnology, Southern Medical University, 3Department of Obstetrics and Gynecology, Chinese PLA General Hospital, 4Clinical Laboratory, Northern Theater Air Force Hospital, 5Guangzhou Darui Reproduction Technology Co., Ltd.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

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Cancer Research

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells
Dongbo Xu 1, Li Wang 1, Kyle Wieczorek 1, Yanqing Wang 2, Xiaojing Zhang 2, David W. Goodrich 2, Qiang Li 1,2
1Department of Urology, Roswell Park Comprehensive Cancer Center, 2Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

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