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Fred Hutchinson Cancer Research Center

16 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay
Tiffany R. Hensley 1, Austin B. Easter 1, Sarah E. Gerdts 1, Stephen C. De Rosa 1, Antje Heit 1, M. Juliana McElrath 1, Erica Andersen-Nissen 1
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.

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JoVE Journal

Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination
Yan Liu 1, Jiayu Liao 1
1Department of Bioengineering, Bourns College of Engineering, University of California, Riverside

A novel method involving quantitative analysis of FRET (Förster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

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Neuroscience

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans
Chao He 1, Jin I. Lee 2, Noelle L'Etoile 3, Damien O'Halloran 1
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.

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Biology

Cryosectioning Yeast Communities for Examining Fluorescence Patterns
Babak Momeni 1, Wenying Shou 1
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.

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Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury
Caramai N. Kamei 1, Yan Liu 1,2, Iain A. Drummond 1,3
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.

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Biology

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors
Jesse J. Hubbard 1, Spencer K. Sullivan 2, Jason A. Mills 3, Brian J. Hayes 1, Beverly J. Torok-Storb 1, Aravind Ramakrishnan 1
1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Division of Hematology, The Children's Hospital of Philadelphia, 3Department 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.

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Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory
Tong Y. Lee 1, Kyoung-hye Yoon 1, Jin I. Lee 1
1Division of Biological Science and Technology, Yonsei University

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

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Genetics

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
Sebastian Grünberg 1, Gabriel E. Zentner 2
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.

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Genetics

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome
Eric J. Foss 1, Uyen Lao 1, Antonio Bedalov 1,2
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.

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Genetics

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome
Vid Leko 1,2, Smitha Sripathy 1, Robin L. Adrianse 1, Taylor Loe 1, Angela Park 1, Uyen Lao 1, Eric J. Foss 1, Marisa S. Bartolomei 3, Antonio Bedalov 1,4
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.

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Developmental Biology

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture
Brandon K. Hadland 1,2, Barbara Varnum-Finney 1, Cynthia Nourigat-Mckay 1, David Flowers 1, Irwin D. Bernstein 1,2
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.

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JoVE Journal

Chemoselective Preparation of 1-Iodoalkynes, 1,2-Diiodoalkenes, and 1,1,2-Triiodoalkenes Based on the Oxidative Iodination of Terminal Alkynes
Youzhi Li *1, Daya Huang *1, Ju Huang 1, Yan Liu 1, Keiji Maruoka 1,2
1School of Chemical Engineering and Light Industry, Guangdong University of Technology, 2Department of Chemistry, Graduate School of Science, Kyoto University

Herein, detailed protocols for the oxidative iodination of terminal alkynes using hypervalent-iodine reagents are presented, which chemoselectively afford 1-iodoalkynes, 1,2-diiodoalkenes, and 1,1,2-triiodoalkenes.

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Genetics

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells
Stefan Radtke 1, Anai M. Perez 1, Rasika Venkataraman 1, Sowmya Reddy 1, Kevin G. Haworth 1, Olivier Humbert 1, Hans-Peter Kiem 1,2,3, Christopher W. Peterson 1,2
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.

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Behavior

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice
Ran You 1,2,3, Yan Liu 3, Raymond Chuen-Chung Chang 3
1Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, 2Nanjing Key Laboratory of Pediatrics, Nanjing Medical University, 3Laboratory of Neurodegenerative Disease, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region (HK SAR)

A comprehensive behavioral test battery of motor skills, mood—including social interaction, depression, and anxiety—and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

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Cancer Research

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices
Nao Nishida-Aoki 1, Andrew J. Bondesson 1,2, Taranjit S. Gujral 1,2,3
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.

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Immunology and Infection

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice
Valerie P. O'Brien 1,2,6, Matthew S. Joens 3,7, Amanda L. Lewis 1,2,4,8, Nicole M. Gilbert 2,5
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.

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