Fred Hutchinson Cancer Research Center View Institution's Website 17 articles published in JoVE Immunology and Infection Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice Valerie P. O'Brien1,2,6, Matthew S. Joens3,7, Amanda L. Lewis1,2,4,8, Nicole M. Gilbert2,5 1Department of Molecular Microbiology, Washington University School of Medicine in Saint Louis, 2Center for Women’s Infectious Disease Research, Washington University School of Medicine in Saint Louis, 3Center for Cellular Imaging, Washington University School of Medicine in Saint Louis, 4Department of Obstetrics and Gynecology, Washington University School of Medicine in Saint Louis, 5Department of Pediatrics, Washington University School of Medicine in Saint Louis, 6Fred Hutchinson Cancer Research Center, 7TESCAN USA, Inc., 8University of California San Diego A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy. Cancer Research Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices Nao Nishida-Aoki1, Andrew J. Bondesson1,2, Taranjit S. Gujral1,2,3 1Division of Human Biology, Fred Hutchinson Cancer Research Center, 2Department of Molecular and Cellular Biology, University of Washington, 3Department of Pharmacology, University of Washington We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions. Genetics Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells Stefan Radtke1, Anai M. Perez1, Rasika Venkataraman1, Sowmya Reddy1, Kevin G. Haworth1, Olivier Humbert1, Hans-Peter Kiem1,2,3, Christopher W. Peterson1,2 1Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, 2Department of Medicine, University of Washington, 3Department of Pathology, University of Washington The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h. Developmental Biology Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture Brandon K. Hadland1,2, Barbara Varnum-Finney1, Cynthia Nourigat-Mckay1, David Flowers1, Irwin D. Bernstein1,2 1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Washington School of Medicine Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors. Genetics G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome Eric J. Foss1, Uyen Lao1, Antonio Bedalov1,2 1Division of Clinical Research, Fred Hutchinson Cancer Research Center, 2Departments of Medicine and Biochemistry, University of Washington We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome. Genetics Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome Vid Leko1,2, Smitha Sripathy1, Robin L. Adrianse1, Taylor Loe1, Angela Park1, Uyen Lao1, Eric J. Foss1, Marisa S. Bartolomei3, Antonio Bedalov1,4 1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 3Epigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 4Departments of Medicine and Biochemistry, University of Washington We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome. Genetics Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae Sebastian Grünberg1, Gabriel E. Zentner2 1Basic Sciences Division, Fred Hutchinson Cancer Research Center, 2Department of Biology, Indiana University We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins. Developmental Biology Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury Caramai N. Kamei1, Yan Liu1,2, Iain A. Drummond1,3 1Nephrology Division, Department of Medicine, Massachusetts General Hospital, 2Basic Sciences Division, Fred Hutchinson Cancer Research Center, 3Department of Genetics, Harvard Medical School Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. Biology Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors Jesse J. Hubbard1, Spencer K. Sullivan2, Jason A. Mills3, Brian J. Hayes1, Beverly J. Torok-Storb1, Aravind Ramakrishnan1 1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Department of Pathology, The Children's Hospital of Philadelphia Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors. Biology Cryosectioning Yeast Communities for Examining Fluorescence Patterns Babak Momeni1, Wenying Shou1 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community. Neuroscience A Molecular Readout of Long-term Olfactory Adaptation in C. elegans Chao He1, Jin I. Lee2, Noelle L'Etoile3, Damien O'Halloran1 1Department of Biological Sciences and Institute for Neuroscience, George Washington University, 2Fred Hutchinson Cancer Research Center, 3Department of Cell and Tissue Biology, University of California San Francisco Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC. Immunology and Infection Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay Tiffany R. Hensley1, Austin B. Easter1, Sarah E. Gerdts1, Stephen C. De Rosa1, Antje Heit1, M. Juliana McElrath1, Erica Andersen-Nissen1 1Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial. Bioengineering Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation Cameron S. Simmons1, Emily Christine Knouf2,3, Muneesh Tewari2,4,5, Lih Y. Lin1 1Electrical Engineering Department, University of Washington, 2Division of Human Biology, Fred Hutchinson Cancer Research Center, 3Molecular and Cellular Biology Program, University of Washington, 4Clinical Research, Fred Hutchinson Cancer Research Center, 5Public Health Sciences, Fred Hutchinson Cancer Research Center Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles. Biology Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry Lei Zhao*1, Jeffrey R. Whiteaker*1, Matthew E. Pope2, Eric Kuhn3, Angela Jackson4, N. Leigh Anderson5, Terry W. Pearson2, Steven A. Carr3, Amanda G. Paulovich1 1Clinical Research Division, Fred Hutchinson Cancer Research Center - FHCRC, 2Department of Biochemistry and Microbiology, University of Victoria, 3Broad Institute of MIT and Harvard, 4Genome BC Proteomics Centre, University of Victoria, 5Plasma Proteome Institute Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format. Biology Generating Chimeric Zebrafish Embryos by Transplantation Hilary A. Kemp1, Amanda Carmany-Rampey1, Cecilia Moens1 1HHMI and Division of Basic Sciences, Fred Hutchinson Cancer Research Center - FHCRC A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage. Biology A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization Bruce W. Draper1, Cecilia B. Moens2 1Molecular and Cellular Biology, University of California, Davis, 2Division of Basic Science, Fred Hutchinson Cancer Research Center - FHCRC This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years. Biology Making Gynogenetic Diploid Zebrafish by Early Pressure Charline Walker1, Greg S. Walsh2, Cecilia Moens2 1Institute of Neuroscience, University of Oregon, 2Division of Basic Science, Fred Hutchinson Cancer Research Center - FHCRC This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.