Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production

Advait Limaye1, Bradford Hall1, Ashok B. Kulkarni1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.13
DOI:  10.1002/0471143030.cb1913s44
Online Posting Date:  September, 2009
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The establishment of mouse embryonic stem (ES) cell lines has allowed for the gene?ration of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene‐targeting experiment. This unit describes detailed step‐by‐step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. Curr. Protoc. Cell Biol. 44:19.13.1‐19.13.24. © 2009 by John Wiley & Sons, Inc.

Keywords: mouse embryonic stem cells; homologous recombination

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Table of Contents

  • Introduction
  • Basic Protocol 1: Introduction of Plasmid DNA into Pluripotent Mouse Embryonic Stem Cells
  • Basic Protocol 2: Selecting, Freezing, and Characterizing Drug‐Resistant ES Cells
  • Support Protocol 1: Culture of Primary Mouse Embryonic Fibroblasts and Preparation of Mouse Embryonic Stem Cell Feeders
  • Support Protocol 2: Culturing Mouse Embryonic Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Introduction of Plasmid DNA into Pluripotent Mouse Embryonic Stem Cells

  • Naïve (non‐electroporated) ES cells growing in culture ( protocol 4) on mitotically arrested feeder layers ( protocol 3) in 6‐cm2 dishes
  • Embryonic stem cell medium (ESM; see recipe)
  • Targeting vector (unit 19.12)
  • Electroporation buffer (EB; see recipe)
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Cellgro)
  • 0.25% (w/v) trypsin/EDTA (Invitrogen)
  • 15‐ and 50‐ml sterile disposable centrifuge tubes
  • Refrigerated centrifuge
  • Electroporation cuvette 0.4‐mm gap (BioRad)
  • Gene Pulser Xcell electroporation system with capacitance extender (BioRad)
  • 250‐ml disposable plastic Erlenmeyer flask, sterile (Corning)
  • 6‐cm2 tissue culture plates of PMEF cells (MMC‐treated; protocol 3)
  • Additional reagents and equipment for restriction enzyme digestion (Bloch and Grossman, ), confirmation of linearized targeting vector (e.g., agarose gel electrophoresis; Voytas, ), ethanol precipitation of DNA (Moore and Dowhan, ), and counting cells (unit 1.1)

Basic Protocol 2: Selecting, Freezing, and Characterizing Drug‐Resistant ES Cells

  • Electroporated ES cells ( protocol 1) growing in 6‐cm2 dishes
  • Embryonic stem cell medium (ESM; see recipe)
  • Positive selection agent (G418; see recipe)
  • Negative selection agent (FIAU; see recipe)
  • Naïve (non‐electroporated) ES cells
  • Calcium‐ and magnesium‐free Dulbecco's PBS (CMF‐DPBS; Cellgro)
  • 0.25% (w/v) trypsin/EDTA (Invitrogen)
  • 2× ES cell freezing medium (see recipe)
  • 0.2% gelatin (from porcine skin, type A; Sigma) in tissue culture‐grade water, sterilized by autoclaving
  • Liquid nitrogen
  • Pipettor from 20 µl to 200 µl to mix cell suspensions, plus sterile tips (Rainin)
  • Pipettor from 200 µl to 1000 µl to mix cell suspensions, plus sterile tips (Rainin)
  • Repeat pipetting device from 100 µl to 1000 µl (useful but optional; Eppendorf)
  • Light‐Touch Pipetting System (Rainin; optional) requires less hand pressure and reduces fatigue
  • 24‐well tissue culture plates (Fisher Scientific)
  • Forceps
  • Flame sterilizer (Fireboy Plus, Integra Biosciences)
  • Glass cloning cylinders in petri dish (see recipe)
  • 2‐ml liquid nitrogen cryovials (Nalgene/Nunc)
  • 12‐well tissue culture plates (Fisher Scientific)
  • Mr. Frosty slow‐cool chamber (Nalgene) containing isopropanol (Fisher Scientific)
  • Liquid nitrogen freezer (Thermo‐Forma)
  • Additional reagents and equipment for preparation of PMEF feeder plates ( protocol 3), purification of DNA by isopropanol precipitation (Laird et al., ), Southern blotting (Brown, ), and PCR ( appendix 3F)

Support Protocol 1: Culture of Primary Mouse Embryonic Fibroblasts and Preparation of Mouse Embryonic Stem Cell Feeders

  • Male mice harboring a positive‐resistance cassette on both alleles (e.g. homozygous knockout mice such as α‐galactosidase A knockout mice obtained from The Jackson Laboratory, Accession no. 003535); and homozygous knockout female mouse
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Cellgro)
  • 70% ethanol
  • 0.05% (w/v) trypsin/EDTA (Invitrogen)
  • Embryonic feeder medium (EFM; see recipe)
  • 2× PMEF cell freezing medium (see recipe)
  • 0.2% gelatin (from porcine skin, type A; Sigma) in tissue culture‐grade water, sterilized by autoclaving
  • Embryonic feeder medium (EFM) containing 10 µg/ml mitomycin C (see recipe)
  • 10‐cm2 and 6‐cm2 tissue culture‐grade dishes (non‐gelatin‐coated; Fisher Scientific)
  • Curved iris scissors (Roboz Surgical Instruments)
  • Straight fine scissors (Roboz Surgical Instruments)
  • Forceps
  • Sterilizer pouches to autoclave instruments (Fine Science Tools)
  • Single‐edged razor blades
  • 50‐ and 15‐ml conical centrifuge tubes
  • Dedicated centrifuge able to achieve 4°C (e.g., Beckman‐Coulter Allegra X‐22R)
  • 2‐ml cryovials (Nalge/Nunc)
  • Mr. Frosty slow‐cool chamber (Nalgene) containing isopropanol (Fisher Scientific)
  • 250‐ml disposable plastic Erlenmeyer flasks, sterile (Corning)
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, ) and counting cells (unit 1.1)

Support Protocol 2: Culturing Mouse Embryonic Stem Cells

  • Embryonic stem cell medium (ESM; see recipe)
  • Pluripotent mouse embryonic stem cells (as frozen stocks; lower passage preferred, usually no more than 20 passages) e.g.:
    • R1 (129 strain–based; ATCC, cat. no. SCRC‐1011)
    • W4129/S6 (129 strain–based; Taconic, cat. no. ES_W412956)
    • Bruce4 (C57BL/6 strain–based; Millipore, cat. no. SF‐CTMI‐2)
    • Pluristem B6 albino (C57BL/6 strain–based; Millipore, cat. no. SCR011; note these cells require IMDM media and 7% CO 2 humidified incubation)
  • Mouse embryonic feeder in 6‐cm2 tissue culture dishes ( protocol 3)
  • 0.25% (w/v) trypsin/EDTA (Invitrogen)
  • 15‐ml centrifuge tubes, sterile
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Literature Cited

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