Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells

Megan D. Hoban1, Zulema Romero1, Gregory J. Cost2, Matthew Mendel2, Michael Holmes2, Donald B. Kohn3

1 Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, 2 Sangamo BioSciences, Richmond, California, 3 Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5B.4
DOI:  10.1002/9780470151808.sc05b04s36
Online Posting Date:  February, 2016
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Abstract

This unit describes the protocol for the delivery of reagents for targeted genome editing to CD34+ hematopoietic stem/progenitor cells (HSPCs). Specifically, this unit focuses on the process of thawing and pre‐stimulating CD34+ HSPCs, as well as the details of their electroporation with in vitro‐transcribed mRNA‐encoding site‐specific nucleases [in this case zinc‐finger nucleases (ZFNs)]. In addition, discussed is delivery of a gene editing donor template in the form of an oligonucleotide or integrase‐defective lentiviral vector (IDLV). Finally, an analysis of cell survival following treatment and downstream culture conditions are presented. While optimization steps might be needed for each specific application with respect to nuclease and donor template amount, adherence to this protocol will serve as an excellent starting point for this further work. © 2016 by John Wiley & Sons, Inc.

Keywords: hematopoietic stem cells; targeted nucleases; electroporation; genome editing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Thawing of CD34+ HSPCs
  • Basic Protocol 2: Electroporation of CD34+ HSPCs
  • Alternate Protocol 1: Addition of Viral Vectors to HSPCs Following Electroporation
  • Basic Protocol 3: Further Culture of Electroporated Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Thawing of CD34+ HSPCs

  Materials
  • Thawing medium (see recipe)
  • CD34+ cells purified from bone marrow, mobilized peripheral blood, or cord blood
  • 1× TD (see recipe)
  • 0.4% Trypan blue
  • Water bath
  • 50‐ml conical centrifuge tubes
  • Centrifuge
  • Hemacytometer
  • 6, 12, or 24‐well non‐tissue culture treated plates
  • Additional reagents and equipment for performing a viable cell count (Phelan, ; unit 1.3)

Basic Protocol 2: Electroporation of CD34+ HSPCs

  Materials
  • CD34+ HSPCs culture dishes (see protocol 1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.4% Trypan blue
  • BTXpress solution (Harvard Apparatus)
  • In vitro‐transcribed mRNA nucleases
  • Oligonucleotides
  • 1× TD medium (see recipe)
  • 50‐ml conical centrifuge tubes
  • Hemacytometer
  • Centrifuge
  • 1.7‐ml RNase/DNase‐free microcentrifuge tubes, sterile
  • 2‐mm gap cuvettes (Harvard Apparatus)
  • BTX ECM 830 Machine (Harvard Apparatus)
  • 24‐well tissue culture‐treated plates
  • Additional reagents and equipment for performing a viable cell count (Phelan, ; unit 1.3)

Alternate Protocol 1: Addition of Viral Vectors to HSPCs Following Electroporation

  Materials
  • Viral vectors
  • 1× TD medium (see recipe)
  • 2× TD medium (see recipe)
  • 1.7‐ml RNase/DNase‐free microcentrifuge tubes, sterile
  • 24‐well tissue culture‐treated plates

Basic Protocol 3: Further Culture of Electroporated Cells

  Materials
  • Cells in culture dishes
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.4% Trypan Blue
  • BBMM medium supplemented with cytokines (see recipe)
  • 1.7‐ml RNase/DNase‐free microcentrifuge tubes, sterile
  • Hemacytometer
  • Centrifuge
  • 24‐well tissue culture‐treated plates
  • Additional reagents and equipment for performing a cell count by trypan blue staining (Phelan, ; unit 1.3)
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Figures

Videos

Literature Cited

Literature Cited
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