Transduction of Herpesvirus saimiri‐Transformed T Cells with Exogenous Genes of Interest

Rubén Martínez‐Barricarte1, Sarah Jill de Jong1, Janet Markle1, Roel de Paus2, Stephanie Boisson‐Dupuis3, Jacinta Bustamante3, Esther van de Vosse2, Bernhard Fleckenstein4, Jean‐Laurent Casanova5

1 St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York, 2 Department of Infectious Diseases, Leiden University Medical Center, Leiden, 3 Paris Descartes University, Imagine Institute, Paris, 4 Institute for Clinical and Molecular Virology, Erlangen‐Nuremberg University, 5 Pediatric Immunology‐Hematology Unit, AP‐HP, Necker Hospital for Sick Children, Paris
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.21C
DOI:  10.1002/cpim.15
Online Posting Date:  November, 2016
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Abstract

Human T cells can be transformed and expanded with herpesvirus saimiri (HVS). HVS‐transformed T cells from patients have facilitated the study of a broad range of primary immunodeficiencies (PID) in which T‐cell development or function is altered. However, the utility of HVS‐transformed T cells for genetic studies has been limited by technical challenges in the expression of exogenous genes, including wild‐type or mutant alleles. A novel, gamma retrovirus–based method for the simple and reliable transduction, purification, and study of HVS‐transformed T cells is described. © 2016 by John Wiley & Sons, Inc.

Keywords: herpesvirus saimiri‐transformed T cells; HVS‐transformed T cell; retrovirus; transduction; T cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Plasmid Construction
  • Basic Protocol 2: Retrovirus Production
  • Basic Protocol 3: Retroviral Transduction of HVS‐Transformed T Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Plasmid Construction

  Materials
  • pLZRS‐IRES‐ΔNGFR plasmid (Fig.  ) (Addgene, deposit no. 72595)
  • Restriction enzyme of choice (New England Biolabs)
  • DpnI enzyme (New England Biolabs)
  • PCR reagents (Denville Scientific)
  • PCR primers for amplifying full‐length gene of interest with an additional 15‐nt tail overlapping target plasmid sequence required for cold fusion reaction
  • Agarose (Invitrogen)
  • Agarose gel extraction kit (Qiagen)
  • Cold Fusion cloning kit (System BioSciences)
  • MAX efficiency Stbl2 competent bacteria (Invitrogen)
  • LB agar
  • Ampicillin (Sigma‐Aldrich)
  • Miniprep kit (Qiagen)
  • Sequencing primers for gene of interest
  • Sanger sequencing reaction reagents (Thermo Fisher Scientific)
  • 37°C water bath
  • Veriti 96‐well plate thermal cycler (Applied Biosystems) or equivalent
  • Nanodrop/spectrophotometer (Thermo Fisher Scientific) or another DNA quantification method/device
  • Bacterial incubator (e.g., Innova42 incubator and shaker, Eppendorf) or equivalent
  • 3730 DNA analyzer (Applied Biosystems) or equivalent Sanger sequencer

Basic Protocol 2: Retrovirus Production

  Materials
  • HVS‐transformed T cells (unit )
  • Dulbecco's phosphate‐buffered saline (DPBS)
  • r‐fibronectin (Takara)
  • Lymphocyte growth medium (see recipe)
  • Concentrated retroviral suspension (see protocol 2)
  • FBS
  • Live/Dead Fixable aqua (Life Technologies)
  • PE‐conjugated anti‐NGFR antibody for flow cytometry (BD Pharmigen)
  • MACS buffer (see recipe)
  • Biotin‐labeled anti‐NGFR antibody (Miltenyi Biotec)
  • Magnetic anti‐biotin microbeads (Miltenyi Biotec)
  • Non‐tissue culture–treated 6‐well plates (Thermo Scientific)
  • Parafilm (Bemis)
  • 5810R centrifuge (Eppendorf) or equivalent
  • Tissue culture incubator (e.g., Heracell 150i incubator, Thermo Scientific or equivalent)
  • Flow cytometer (e.g., LSRII, BD or any other cytometer with violet and yellow/green lasers)
  • 25‐cm tissue‐culture flasks (Falcon)
  • MS columns (Miltenyi Biotec)
  • OctoMACS separator (Miltenyi Biotec)
  • 15‐ml centrifuge tubes (Falcon)
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Figures

Videos

Literature Cited

Literature Cited
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