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Beckman Research Institute of City of Hope

6 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery
Jiehua Zhou 1, Haitang Li 1, Jane Zhang 2, Swiderski Piotr 3, John Rossi 1
1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 2Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Shared Resource-DNA/RNA Peptide, Beckman Research Institute of City of Hope

Several 2’-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

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Biology

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors
Michael Winkler 1, Nancy Trieu 2, Tao Feng 2, Liang Jin 2, Stephanie Walker 2, Lipi Singh 2, Hsun Teresa Ku 2,3
1Department of Biotechnology & Bioinformatics, California State University Channel Islands, 2Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, 3The Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

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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 Luo 1, Janine C. Quijano 1, Lena Wedeken 1, Kevin Jou 1, Arthur D. Riggs 1, David A. Tirrell 3, H. Teresa Ku 1
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.

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Immunology and Infection

Humanized NOD/SCID/IL2rγnull (hu-NSG) Mouse Model for HIV Replication and Latency Studies
Xin Xia 1, Haitang Li 1, Sangeetha Satheesan 1,2, Jiehua Zhou 1, John J. Rossi 1
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.

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Immunology and Infection

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry
Jerlisa Ann C. Arizala 1,2, Pritsana Chomchan 1, Haitang Li 1, Roger Moore 3, Helen Ge 3, Dominique L. Ouellet 1, John J. Rossi 1,2
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.

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Genetics

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells
Takahiro Yamakawa 1, Keiichi Itakura 1
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.

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