Generation of Human Embryonic Stem Cell Reporter Knock‐In Lines by Homologous Recombination

Richard P. Davis1, Catarina Grandela2, Koula Sourris3, Tanya Hatzistavrou3, Mirella Dottori4, Andrew G. Elefanty3, Edouard G. Stanley3, Magdaline Costa3

1 Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands, 2 Laboratory of Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Academic Medical Center, The Netherlands, 3 Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Australia, 4 Centre for Neuroscience and Dept of Pharmacology, The University of Melbourne, Parkville, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5B.1
DOI:  10.1002/9780470151808.sc05b01s11
Online Posting Date:  November, 2009
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Abstract

This unit describes a series of technical procedures to form clonal human embryonic stem cell (hESC) lines that are genetically modified by homologous recombination. To develop a reporter knock‐in hESC line, a vector is configured to contain a reporter gene adjacent to a positive selection cassette. These core elements are flanked by homologous sequences that, following electroporation into hESCs, promote the integration of the vector into the appropriate genomic locus. The positive selection cassette facilitates the enrichment and isolation of genetically modified hESC colonies that are then screened by PCR to identify correctly targeted lines. The selection cassette, flanked by loxP sites, is subsequently excised from the positively targeted hESCs via the transient expression of Cre recombinase. This is necessary because the continued presence of the cassette may interfere with the regulation of the reporter or neighboring genes. Finally, these genetically modified hESCs are clonally isolated using single‐cell deposition flow cytometry. Reporter knock‐in hESC lines are valuable tools that allow easy and rapid identification and isolation of specific hESC derivatives. Curr. Protoc. Stem Cell Biol. 11:5B.1.1‐5B.1.34. © 2009 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; hESCs; gene targeting; homologous recombination; fluorescent reporter gene

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Electroporation of hESCs and Selection of Antibiotic‐Resistant hESCs
  • Support Protocol 1: Picking and Expanding Antibiotic‐Resistant hESC Colonies
  • Support Protocol 2: Preparation of Genomic DNA from hESCs Growing in 48‐Well Tissue Culture Plates
  • Alternate Protocol 1: Isolation of Genomic DNA from hESCs Using Phenol/Chloroform Extraction
  • Support Protocol 3: PCR Identification of Targeted hESC Clones
  • Basic Protocol 2: Removing the Positive Selection Cassette from Genetically Modified hESCs by Transient Expression of Cre Recombinase
  • Support Protocol 4: Clonal Isolation of hESCs by Single‐Cell Deposition Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Electroporation of hESCs and Selection of Antibiotic‐Resistant hESCs

  Materials
  • hESCs in 150‐cm2 tissue culture flasks at enzymatic passage 5 to 10 (see unit 1.1) in hESC medium (see recipe)
  • 150‐cm2 gelatinized tissue culture flasks (see recipe) preseeded with mitotically inactivated MEFs at 1 × 104/cm2 for passaging hESCs prior to electroporation
  • MEF medium (see recipe)
  • Trypsin (see recipe) or TrypLE Select cell dissociation enzyme (Invitrogen)
  • hESC medium (see recipe), 37°C
  • Phosphate‐buffered saline without CaCl 2 and MgCl 2 (CMF‐PBS; Invitrogen)
  • 0.4% (w/v) Trypan blue (Fluka)
  • Soybean trypsin inhibitor (see recipe; Invitrogen), optional
  • Linearized targeting vector (see ) in Tris/EDTA (TE) buffer (see recipe) for transfection
  • 60‐mm gelatinized tissue culture dishes preseeded with 2 × 104/cm2 mitotically inactivated MEFs
  • Geneticin/G418 Selective Antibiotic (Invitrogen)
  • Mitotically inactivated, irradiation‐treated (unit 1.3) G418‐resistant mouse embryonic fibroblasts (MEFs; Conner, )
  • 37°C water bath
  • Gene Pulser cuvette, 0.4‐cm electrode gap, sterile (Bio‐Rad, cat. no. 165‐2088)
  • 15‐ and 50‐ml sterile centrifuge tubes
  • Refrigerated centrifuge
  • Gilson pipettors (John Morris Scientific) or equivalent, with sterile (plugged) tips
  • Tissue culture microscope with phase contrast objectives and phase rings
  • Hemacytometer (Neubauer)
  • Electroporator (Gene Pulser II System; Bio‐Rad)
  • Sterile Pasteur pipets

Support Protocol 1: Picking and Expanding Antibiotic‐Resistant hESC Colonies

  Materials
  • Flat‐bottomed 48‐well tissue culture plates, gelatinized (see recipe) and preseeded with mitotically inactivated MEFs at 2 × 104/cm2
  • Mitotically inactivated, irradiation‐treated (unit 1.3) mouse embryonic fibroblasts (MEFs; Conner, )
  • MEF medium (see recipe)
  • hESC medium (see recipe)
  • 60‐mm tissue culture dishes containing drug‐resistant hESC colonies from either protocol 1 or protocol 62
  • 26‐G, ½‐in. (0.45 × 13–mm) needles
  • 1‐ml syringe
  • 200‐µl pipet tips
  • Gilson pipettors (John Morris Scientific) or equivalent, with sterile (plugged) tips
  • Stereomicroscope

Support Protocol 2: Preparation of Genomic DNA from hESCs Growing in 48‐Well Tissue Culture Plates

  Materials
  • 48‐well tissue culture plates labeled “DNA,” containing confluent hESC colonies (from protocol 2, step 13)
  • Phosphate‐buffered saline without CaCl 2 and MgCl 2 (CMF‐PBS; Invitrogen)
  • DNA lysis buffer containing 200 µg/ml proteinase K (see recipe)
  • 100% (v/v) and 70% (v/v) ethanol
  • Tris/EDTA buffer (TE buffer; see recipe)
  • Temperature‐adjustable incubator
  • Centrifuge with multi‐well plate spinner attachment
  • Blotting paper
  • Gilson pipettors (John Morris Scientific) or equivalent, with sterile (plugged) tips

Alternate Protocol 1: Isolation of Genomic DNA from hESCs Using Phenol/Chloroform Extraction

  Materials
  • 48‐well tissue culture plates containing confluent hESC colonies ( protocol 2, step 13) or microcentrifuge tubes containing clumps of hESCs ( protocol 2, step 13, annotation)
  • DNA lysis buffer containing 200 µg/ml proteinase K (see recipe)
  • Phenol/chloroform/isoamyl alcohol (25:24:1) solution saturated with 10 mM Tris⋅Cl, pH 8.0/1 mM EDTA
  • 70 %(v/v) and 100% (v/v) ethanol
  • Trypsin/EDTA buffer (TE buffer; see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Microcentrifuge
  • 55°C incubator

Support Protocol 3: PCR Identification of Targeted hESC Clones

  Materials
  • PCR master mix (see Table 5.1.2) containing:
    • Autoclaved, distilled water
    • 10× High Fidelity PCR Buffer (Invitrogen)
    • 10 mM dNTP mixture (Sigma)
    • 50 mM MgSO 4
    • Forward & Reverse Primers (see Table 5.1.1)
    • DMSO
    • Platinum Taq High Fidelity DNA polymerase (Invitrogen)
  • Genomic DNA from the hESC clones ( protocol 3 or the protocol 4)
  • hESC medium (see recipe)
  • Mitotically inactivated, irradiation‐treated (unit 1.3) mouse embryonic fibroblasts (MEFs; Conner, )
  • MEF medium (see recipe)
  • 0.2‐ml nuclease‐free PCR tubes
  • 1.5‐ml nuclease‐free microcentrifuge tubes
  • Gilson pipettors (John Morris Scientific) or equivalent, with sterile (plugged) tips
  • Microcentrifuge
  • DNA thermal cycler
  • Gelatinized organ culture dishes
  • Additional reagents and equipment for analyzing the amplification products by agarose gel electrophoresis (Voytas, ) and maintaining and expanding hESCs by both mechanical and enzymatic passaging (unit 1.1)

Basic Protocol 2: Removing the Positive Selection Cassette from Genetically Modified hESCs by Transient Expression of Cre Recombinase

  Materials
  • Gene‐targeted hESCs containing a loxP‐flanked positive selection cassette in gelatinized 75‐cm2 tissue culture flasks between enzymatic passages 5 and 10 co‐cultured with MEFs pre‐seeded at a density of 1.5 × 106/75‐cm2 flask
  • Phosphate‐buffered saline without CaCl 2 and MgCl 2 (CMF‐PBS; Invitrogen)
  • Trypsin (see recipe) or TrypLE Select cell dissociation enzyme (Invitrogen)
  • hESC medium (see recipe)
  • 60‐mm gelatinized tissue culture dishes (see recipe) seeded with 3 × 104/cm2 mitotically inactivated MEFs
  • Mitotically inactivated, irradiation‐treated (unit 1.3) mouse embryonic fibroblasts (MEFs; Conner, )
  • FuGENE 6 Transfection Reagent (Roche)
  • DMEM/F12 (Invitrogen)
  • pEFBOS‐CreIRESpuro expression vector (GenBank accession number EU693012; available on request from the authors' laboratory; e‐mail request to andrew.elefanty@med.monash.edu.au or ed.stanley@med.monash.edu.au)
  • Puromycin solution (Sigma), 10 mg/ml
  • Gilson pipettors (John Morris Scientific) or equivalent, with sterile (plugged) tips
  • 37°C incubator
  • 15‐ and 50‐ml sterile centrifuge tubes
  • Refrigerated centrifuge
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for performing a cell count (Phelan, ; unit 1.3), transferring colonies to 48‐well tissue culture plates ( protocol 2), extracting DNA from colonies ( protocol 3), and screening extracted DNA by PCR ( protocol 5)

Support Protocol 4: Clonal Isolation of hESCs by Single‐Cell Deposition Flow Cytometry

  Materials
  • 0.1% (w/v) gelatin solution (see recipe)
  • Mitotically inactivated, irradiation‐treated (unit 1.3) mouse embryonic fibroblasts (MEFs; Conner, )
  • MEF medium (see recipe)
  • Genetically modified hESCs generated from protocol 6 in 75‐cm2 tissue culture flasks between enzymatic passages 5 and 10
  • 75‐cm2 gelatinized tissue culture flasks (see recipe) seeded with mitotically inactivated MEFs at 1 × 104/cm2 for passaging hESCs prior to cloning
  • hESC medium (see recipe)
  • Recombinant human FGF2 (see recipe)
  • Phosphate‐buffered saline without CaCl 2 and MgCl 2 (CMF‐PBS; Invitrogen)
  • Trypsin (see recipe) or TrypLE Select cell dissociation enzyme (Invitrogen)
  • Propidium iodide (PI) solution (see recipe)
  • Flat‐bottomed 48‐well tissue culture plates, gelatinized and seeded with mitotically inactivated MEFs at 0.75 × 106/plate
  • Liquid nitrogen
  • 96‐well flat‐bottom tissue culture‐treated plates and lids
  • 37°C, 5% CO 2 incubator
  • 15‐ml sterile centrifuge tubes
  • Refrigerated centrifuge
  • 5‐ml sterile round‐bottom polystyrene FACS tubes (12 × 75–mm) with 35‐µm cell‐strainer caps and with snap lids (Falcon)
  • Parafilm M (Pechiney Plastic Packaging) or equivalent
  • Flow cytometer with single‐cell deposition function, e.g., FACSVantageSE‐DiVa system (Becton Dickinson) or equivalent
  • Inverted microscope
  • Gilson pipettors (John Morris Scientific) or equivalent, with sterile (plugged) tips
  • Stereomicroscope
  • Additional reagents and equipment for propagating hESCs in bulk culture (unit 1.3), extracting DNA from colonies ( protocol 3), screening extracted DNA by PCR ( protocol 5), maintaining and expanding the genetically modified lines (unit 1.1), and for cryopreserving hESCs (Reubinoff et al., )
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Figures

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Literature Cited

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