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Broad Institute

15 ARTICLES PUBLISHED IN JoVE

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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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Immunology and Infection

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
Rachel Daniels 1,2, Elizabeth J. Hamilton 2, Katelyn Durfee 2, Daouda Ndiaye 3, Dyann F. Wirth 2,5, Daniel L. Hartl 1, Sarah K. Volkman 2,4
1Department of Organismic and Evolutionary Biology, Harvard University, 2Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 3Faculty of Medicine and Pharmacy, Cheikh Anta Diop University, 4School of Nursing and Health Sciences, Simmons College, 5Institute of Infectious Diseases, Broad Institute

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

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Immunology and Infection

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Savannah E. Sanchez 1, Daniel A. Cuevas 2, Jason E. Rostron 1, Tiffany Y. Liang 3, Cullen G. Pivaroff 1, Matthew R. Haynes 1, Jim Nulton 4, Ben Felts 4, Barbara A. Bailey 4, Peter Salamon 4, Robert A. Edwards 1,5,6, Alex B. Burgin 7, Anca M. Segall 1, Forest Rohwer 1
1Department of Biology, San Diego State University, 2Computational Science Research Center, San Diego State University, 3Bioinformatics and Medical Informatics Research Center, San Diego State University, 4Department of Mathematics and Statistics, San Diego State University, 5Department of Computer Science, San Diego State University, 6Mathematics and Computer Science Division, Argonne National Laboratory, 7SPARC Committee, Broad Institute

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

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Biology

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
Leanna S. Morinishi 1, Paul Blainey 2
1Broad Institute, 2Department of Biological Engineering, Massachusetts Institute of Technology

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

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Biology

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis
Alexandra Naba 1, Karl R. Clauser 2, Richard O. Hynes 1
1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Proteomics Platform, Broad Institute

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

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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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Cancer Research

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells
Nouran S. Abdelfattah 1, Ann Mullally 1,2,3
1Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 2Broad Institute, 3Dana-Farber Cancer Institute, Harvard Medical School

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

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JoVE Journal

CRISPR Guide RNA Cloning for Mammalian Systems
Sathiji Nageshwaran *1,2, Alejandro Chavez *1,2,3, Nan Cher Yeo 1,2, Xiaoge Guo 1,2, Alissa Lance-Byrne 1, Angela Tung 1, James J. Collins 1,4,5,6,7, George M. Church 1,2
1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Genetics, Harvard Medical School, 3Department of Pathology, Massachusetts General Hospital, 4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 5Synthetic Biology Center, Massachusetts Institute of Technology, 6Department of Biological Engineering, Massachusetts Institute of Technology, 7Broad Institute

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

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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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Biology

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury
Hussein Atta *1,2, Ali Hashemi Gheinani *1,2, Amanda Wacker 1, Yaser Heshmati 3,4,5, Alex Bigger-Allen 1,6, George Lambrinos 1,2, Yao Gao 2,7, Diane R. Bielenberg 2,7, Rosalyn M. Adam 1,2
1Department of Urology, Boston Children's Hospital, 2Department of Surgery, Harvard Medical School, 3Division of Hematology/Oncology, Harvard Medical School Boston, 4Dana-Farber Cancer Institute, 5Broad Institute, 6Biological Biomedical Sciences Program, Division of Medical Sciences, Harvard Medical School, 7Vascular Biology Program, Boston Children's Hospital

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

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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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