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Dana-Farber Cancer Institute

12 ARTICLES PUBLISHED IN JoVE

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Biology

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures
Maria F. Pazyra-Murphy 1,2, Rosalind A. Segal 1,2
1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

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Bioengineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture
Christopher Moraes 1,2, Yu Sun 1,2, Craig A. Simmons 1,2,3
1Department of Mechanical and Industrial Engineering, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Faculty of Dentistry, University of Toronto

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

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Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells
Susan Fotheringham 1,2, Keren Levanon 1,2,3, Ronny Drapkin 1,2,4
1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

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JoVE Journal

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
Daniel E. Bauer *1,2,3, Matthew C. Canver *1, Stuart H. Orkin 1,2,3,4
1Harvard Medical School, 2Division of Hematology/Oncology, Boston Children's Hospital, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, 4Howard Hughes Medical Institute

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

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Medicine

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies
Emily J. Chadwick 1, David P. Yang 1, Mariella G. Filbin 2, Emanuele Mazzola 3, Yu Sun 1, Oded Behar 1,4, Maria F. Pazyra-Murphy 1, Liliana Goumnerova 5, Keith L. Ligon 6, Charles D. Stiles 1, Rosalind A. Segal 1
1Department of Cancer Biology, Dana-Farber Cancer Institute, 2Department of Pediatrics, Children's Hospital, 3Department of Biostatistics & Computational Biology, Dana-Farber Cancer Institute, 4Department of Developmental Biology and Cancer Research, Hebrew University of Jerusalem, 5Department of Neurosurgery, Children's Hospital, 6Center for Molecular Oncologic Pathology, Department of Medical Oncology, Dana-Farber Cancer Institute

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice.  

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Biology

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Erik Serrao 1, Peter Cherepanov 2, Alan N. Engelman 1
1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 2Chromatin Structure and Mobile DNA, The Francis Crick Institute

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

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Developmental Biology

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae
Elliott J. Hagedorn 1,2, Jennifer L. Cillis 3, Caitlyn R. Curley 3, Taylor C. Patch 3, Brian Li 1,2, Bradley W. Blaser 1,2,7, Raquel Riquelme 1,2, Leonard I. Zon 1,2,4,5,6, Dhvanit I. Shah 1,2,3,4,5
1Division of Hematology/Oncology, Boston Children’s Hospital, 2Harvard Medical School, 3Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

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Developmental Biology

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Charles A. C. Williams 1, Nathanael S. Gray 2,3, Greg M. Findlay 1
1The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2Department of Cancer Biology, Dana-Farber Cancer Institute, 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition.

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Cancer Research

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Erin R. Bonner 1,2, Karim Saoud 1, Sulgi Lee 1,2, Eshini Panditharatna 3, Madhuri Kambhampati 1, Sabine Mueller 4,5, Javad Nazarian 1,2,5
1Center for Genetic Medicine, Children's National Health System, 2Institute for Biomedical Sciences, The George Washington University School of Medicine and Health Sciences, 3Dana-Farber Cancer Institute, 4Department of Neurology, University of California San Francisco, 5DIPG Centre of Expertise Zurich, Universitats-Kinderspital Zurich

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

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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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Cancer Research

ATAC-Seq Optimization for Cancer Epigenetics Research
Mikhala Cooper *1, Atrayee Ray *1, Atrayee Bhattacharya 2, Archana Dhasarathy 1, Motoki Takaku 1
1University of North Dakota School of Medicine and Health Sciences, 2Dana-Farber Cancer Institute

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

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Small Molecule Screening Strategies from Lead Identification to Validation
Milka Kostic 1
1Department of Cancer Biology, Chemical Biology Program, Dana-Farber Cancer Institute

Small Molecule Screening Strategies from Lead Identification to Validation

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