TLX1 (HOX11) Immortalization of Embryonic Stem Cell–Derived and Primary Murine Hematopoietic Progenitors

Robert G. Hawley1, Teresa S. Hawley2, Alan B. Cantor3

1 The George Washington University Medical Center, Washington, D.C., 2 Flow Cytometry Core Facility, The George Washington University Medical Center, Washington, D.C., 3 Dana‐Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.7
DOI:  10.1002/9780470151808.sc01f07s7
Online Posting Date:  December, 2008
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The ability to generate genetically engineered cell lines is of great experimental value. They provide a renewable source of material that may be suitable for biochemical analyses, chromatin immunoprecipitation assays, structure‐function studies, gene function assignment, and transcription factor target gene identification. This unit describes protocols for TLX1 (HOX11)‐mediated immortalization of murine hematopoietic progenitors derived from in vitro differentiated murine embryonic stem cells, or from primary mouse fetal liver or bone marrow. A wide variety of hematopoietic cell types have been immortalized using these procedures including erythroid, megakaryocytic, monocytic, myelocytic, and multipotential cell types. These lines are typically cytokine dependent for their survival and growth. Curr. Protoc. Stem Cell Biol. 7:1F.7.1‐1F.7.19. © 2008 by John Wiley & Sons, Inc.

Keywords: murine cell immortalization; TLX1; HOX11; hematopoietic in vitro ES cell differentiation; hematopoietic progenitors

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Table of Contents

  • Introduction
  • Basic Protocol 1: TLX1 Immortalization of ES Cell In Vitro Differentiated Hematopoietic Progenitors
  • Alternate Protocol 1: TLX1 Immortalization of Primary Murine Hematopoietic Cells
  • Support Protocol 1: Maintenance of TLX1 (HOX11) Retroviral Producer Cell Lines
  • Support Protocol 2: Cloning of Immortalized Cells in Methylcellulose
  • Support Protocol 3: Cryopreservation of Immortalized Cells
  • Support Protocol 4: Immunophenotypic Analysis of Immortalized Cell Lines by FACS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: TLX1 Immortalization of ES Cell In Vitro Differentiated Hematopoietic Progenitors

  • Murine ES cells, gel‐adapted (grown on gelatinized plates, not on feeder cells) and of low passage number (see unit 1.4)
  • IMDM‐ES‐15 with MTG, LIF, and pen/strep (see recipe)
  • 0.05% (w/v) and 0.25% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25200 and 25300, respectively)
  • IMDM‐ES‐5 with MTG and pen/strep (see recipe)
  • Primary ES cell differentiation medium (see recipe)
  • TLX1‐retroviral producer cell line (see Support Protocol):
    • GP+E‐86/MSCV‐HOX11 for cotransduction of the neomycin phosphotransferase (neo) gene conferring resistance to the neomycin analog Geneticin in mammalian cells or
    • GP+E‐86/MSCVhyg‐HOX11 for cotransduction of the hygromycin B phosphotransferase (hyg) drug resistance gene
  • Isocove's modified Dulbecco's medium (IMDM; Invitrogen, cat. no. 12440)
  • IMDM‐ES‐15: IMDM supplemented with 15% (v/v) ES‐FBS
  • Coculture medium (see recipe)
  • IMDM‐ES‐15 with glutamine and pen/strep (see recipe)
  • Immortalized cell medium (see recipe)
  • 50 mg/ml Geneticin (Invitrogen cat. no. 10131‐035) or 50 mg/ml hygromycin B (Mediatech) stock
  • Gelatinized 25‐cm2 tissue culture flasks (see recipe)
  • 15‐ml and 50‐ml conical centrifuge tubes (sterile)
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 100‐mm petri dishes
  • 100‐mm and 60‐mm tissue culture dishes
  • 20‐G needle and 3‐ml syringe
  • Hemacytometer
  • Cell irradiator (optional)
  • Additional reagents and equipment for counting viable cells (unit 1.3)

Alternate Protocol 1: TLX1 Immortalization of Primary Murine Hematopoietic Cells

  • Adult mouse
  • Mouse dissection tools
  • 70% ethanol spray solution
  • 1.5‐ml microcentrifuge tubes, sterile
  • 1× PBS, sterile: diluted from 10× phosphate‐buffered saline without calcium and magnesium (Sigma, cat. no. P7059) to 1× with water, and sterilized by passing through a 0.22‐µm filter
  • 70‐µm cell strainer (BD Falcon; optional)
  • 22‐G needle and 1‐ml syringe (for bone marrow progenitors)
  • 23‐G needle and 1‐ml syringe (for fetal liver progenitors)
  • 25‐G needle and 1‐ml syringe (for fetal liver progenitors)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 1: Maintenance of TLX1 (HOX11) Retroviral Producer Cell Lines

  • Suspension of immortalized cells ( protocol 1 or protocol 2)
  • Methylcellulose medium (see recipe)
  • Immortalized cell medium (see recipe), containing the appropriate selection antibiotic
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 15‐ml centrifuge tubes (sterile)
  • 5‐ml syringe fitted with a 16‐G needle
  • 35‐mm tissue culture dish (Falcon, cat. no. 35‐1008)
  • 150‐mm bacterial dishes (Falcon, cat. no. 35‐1058)
  • Inverted microscope
  • 2‐ to 20‐µl micropipettor and sterile, disposable tips
  • 96‐well flat bottom tissue culture plate (Costar, cat. no. 3596)
  • 24‐well tissue culture plate (Falcon, cat. no. 35‐3047)
  • Additional reagents and equipment for counting cells (unit 1.3)

Support Protocol 2: Cloning of Immortalized Cells in Methylcellulose

  • Suspension of immortalized cells ( protocol 1, protocol 2, or protocol 4)
  • Immortalized cell medium (see recipe), ice cold
  • 2× freezing medium (see recipe), ice cold
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 15‐ml centrifuge tubes (sterile)
  • 1.8‐ml cryovials (Nunc, cat. no. 377267)
  • −80°C freezer
  • Liquid nitrogen storage tank

Support Protocol 3: Cryopreservation of Immortalized Cells

  • Cell line suspensions ( protocol 1 or protocol 2)
  • FACS wash buffer (see recipe)
  • Fluorochrome‐conjugated antibodies and isotype matched control antibodies (see Table 1.7.1); choice of fluorochromes depends on the flow cytometer being used and the combination of markers being analyzed
  • Tissue culture centrifuge (refrigerated, benchtop centrifuge with swinging bucket rotor)
  • 15‐ml conical centrifuge tube
  • 12 × 75–mm FACS tubes (BD Falcon, cat. no. 35‐2054)
  • Flow cytometer (FACS analyzer)
  • Additional reagents and equipment for counting cells (unit 1.3)
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