Beckman Research Institute of City of Hope 5 articles published in JoVE Immunology and Infection Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry Jerlisa Ann C. Arizala1,2, Pritsana Chomchan1, Haitang Li1, Roger Moore3, Helen Ge3, Dominique L. Ouellet1, John J. Rossi1,2 1Molecular and Cellular Biology Department, Beckman Research Institute at the City of Hope, 2Irell & Manella Graduate School of Biological Sciences, 3Department of Molecular Immunology, Beckman Research Institute and the City of Hope Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways. Genetics Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells Takahiro Yamakawa1, Keiichi Itakura1 1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion. Immunology and Infection Humanized NOD/SCID/IL2rγnull (hu-NSG) Mouse Model for HIV Replication and Latency Studies Xin Xia1, Haitang Li1, Sangeetha Satheesan1,2, Jiehua Zhou1, John J. Rossi1 1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 2Irell and Manela Graduate School of Biological Sciences, Beckman Research Institute of City of Hope This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations. Developmental Biology In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas Jacob R. Tremblay*1,2, Jeanne M. LeBon*1, Angela Luo1, Janine C. Quijano1, Lena Wedeken1, Kevin Jou1, Arthur D. Riggs1, David A. Tirrell3, H. Teresa Ku1 1Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope, 2Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Division of Chemistry and Chemical Engineering, California Institute of Technology In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described. Bioengineering An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles Grace Y. Tang1, Melanie A. Pribisko1, Ryan K. Henning1, Punnajit Lim2, John Termini2, Harry B. Gray1, Robert H. Grubbs1 1Division of Chemistry and Chemical Engineering, California Institute of Technology, 2Department of Molecular Medicine, Beckman Research Institute of the City of Hope Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.
