Gene Editing in One‐Cell Embryos by Zinc‐Finger and TAL Nucleases

Benedikt Wefers1, Melanie Meyer2, Svenja Hensler2, Sudeepta Panda2, Oskar Ortiz1, Wolfgang Wurst3, Ralf Kühn2

1 Helmholtz Center Munich, Institute for Developmental Genetics Munich, Germany, 2 Chair for Developmental Genetics, Technische Universität München, Munich, Germany, 3 Deutsches Zentrum für Neurodegenerative Erkrankungen, Standort München, Munich, Germany
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo120177
Online Posting Date:  December, 2012
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Abstract

Gene targeting by sequence‐specific nucleases in one‐cell embryos provides an expedited mutagenesis approach in rodents. This technology has been recently established to create knockout and knockin mutants through sequence deletion or sequence insertion. This article provides protocols for the preparation and microinjection of nuclease mRNA and targeting vector DNA into fertilized mouse eggs. Furthermore, we provide guidelines for genotyping the desired mouse mutants. Curr. Protoc. Mouse Biol. 2:347‐364 © 2012 by John Wiley & Sons, Inc.

Keywords: pronucleus injection; gene targeting; mouse mutant; zinc‐finger nuclease; TAL nuclease; homologous recombination

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of T10E0.1 Buffer for Pronucleus Injection
  • Basic Protocol 2: Preparation of Nuclease mRNA
  • Basic Protocol 3: Preparation of Gene‐Targeting Vector and Oligonucleotide DNA
  • Basic Protocol 4: Preparation of mRNA/DNA Aliquots for Embryo Injection
  • Basic Protocol 5: Microinjection of One‐Cell Embryos
  • Basic Protocol 6: Genotyping of Nuclease‐Induced Mutants
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of T10E0.1 Buffer for Pronucleus Injection

  Materials
  • Trizma base (Sigma, cat. no. T6791)
  • Trizma hydrochloride (Sigma, cat. no. T6666)
  • H 2O, embryo‐tested (Sigma, cat. no. W1503)
  • Disodium EDTA (Sigma, cat. no. E4884)
  • 50‐ml conical polypropylene centrifuge tubes (BD Falcon)
  • pH test strips, pH 4.5‐ 10
  • Syringe filter unit Millex GV, 0.22 µm (Millipore, cat. no. SLGV033RS)

Basic Protocol 2: Preparation of Nuclease mRNA

  Materials
  • Plasmid DNAs containing nuclease coding regions (e.g., ZFN coding pVax plasmids from Sigma‐Aldrich)
  • Restriction enzyme that cuts once downstream of the nuclease stop codon of the plasmid (pVax plasmids can be linearized by XbaI digestion)
  • 0.8% agarose gel (Voytas, )
  • Spin‐column gel‐extraction kit (Qiagen)
  • 3 M sodium acetate, pH 5.2
  • 70% and 100% ethanol
  • RNase‐free H 2O
  • Message Machine T7 Ultra kit (Ambion, cat. no. AM1345)
  • MegaClear kit (Ambion, cat. no. AM1908)
  • T 10E 0.1 injection buffer ( protocol 1)
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • NorthernMax‐Gly kit (Ambion, cat. no. AM1946)
  • Millenium RNA size marker (Ambion, cat. no. AM7150)
  • 50° and 65°C water baths or heat block
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) and spectrophotometric determination of DNA and RNA (Gallagher, )

Basic Protocol 3: Preparation of Gene‐Targeting Vector and Oligonucleotide DNA

  Materials
  • Plasmid preparation of gene‐targeting vector, or synthetic DNA oligonucleotide
  • 3 M sodium acetate, pH 5.2
  • 100% ethanol
  • 70% ethanol (prepared with embryo‐tested H 2O)
  • Water, embryo tested (Sigma, cat. no. W1503)
  • T 10E 1 injection buffer (see recipe)
  • Oligonucleotide pellet (molecules of ∼140 nucleotide length; Metabion; http://www.metabion.com/)
  • MF Membrane Filter 0.025 µm VSWP (Millipore, cat no. VSWP02500)
  • 10‐cm tissue culture dish
  • Additional reagents and equipment for spectrophotometric determination of DNA (Gallagher, )

Basic Protocol 4: Preparation of mRNA/DNA Aliquots for Embryo Injection

  Materials
  • Purified nuclease mRNAs in injection buffer ( protocol 2, step 26)
  • T 10E 0.1 injection buffer ( protocol 1)
  • Purified vector DNA in injection buffer ( protocol 3)
  • New bag of 1.5‐ml microcentrifuge tubes (Eppendorf)

Basic Protocol 5: Microinjection of One‐Cell Embryos

  Materials
  • 15 female mice for superovulation [FVB inbred or (C57BL/6× DBA/2)F 1 hybrid strain, 4 weeks of age]
  • PMSG (pregnant mare serum gonadotropin; e.g., Sigma, cat. no. G4877); prepare a stock solution of 50 IU/ml with sterile H 2O and store in aliquots at −20°C up to 2 months
  • hCG (human chorionic gonadotropin; e.g., Sigma, cat. no. CG5); prepare a stock solution of 50 IU/ml with sterile H 2O and store in aliquots at −20°C up to 2 months
  • 15 male mice for mating
  • M2 medium (embryo tested, Sigma, cat. no. M7167)
  • Hyaluronidase (type IV‐S from bovine testes, embryo tested, Sigma, cat. no. H4272); prepare a stock solution of 10 mg/ml in M2 medium, filter sterilize, and store up to 6 months at −20°C in single‐use aliquots of 50 µl
  • Injection solution ( protocol 4)
  • Pseudopregnant female mice (see annotation to step 25)
  • 6‐cm bacteriological petri dishes
  • Inverted stereomicroscope [e.g., Leica DMI3000B with 40× 0.55 Corr Ph2 objective, phase contrast, differential interference contrast (DIC), and polarizer]
  • Fine forceps
  • Transfer pipets (e.g., HB 1.80 × 1.20, L=160 mm, 125‐134 µm, BW=45°, BL=10 mm; BioMedical Instruments, http://biomedical‐instruments.de)
  • Gel loading pipet tips (e.g., Eppendorf Microloader tips)
  • Pronucleus injection capillaries (e.g., BM100F‐10, end fire‐polished, PI‐1.6, Barnow; BioMedical Instruments, http://bio‐medical.com/; injection capillaries must be of the “filament” type to enable their filling with fluid)
  • Microinjector device for application of positive and negative pressure (e.g., Eppendorf FemtoJet)
  • Depression (concavity) slides (e.g., Electron Microscopy Sciences, cat. no. 71878‐01)
  • Holding pipets (e.g., BM100T‐15, broad, ID = 20‐25 µm, straight; BioMedical Instruments)
  • Additional reagents and equipment for sacrifice of mice (Donovan and Brown, ) and embryo transfer (Nagy et al., )

Basic Protocol 6: Genotyping of Nuclease‐Induced Mutants

  Materials
  • PCR primers (to amplify a region of ∼250 bp; sequence depends on target region)
  • PCR amplification reagents (e.g., 5‐Prime MasterMix, 5 Prime, cat. no. 2200100; also see Kramer and Coen, )
  • Tail DNA (for a protocol, see Nagy et al., ) from founder pups ( protocol 5) and a wild‐type control
  • Restriction enzymes (again, depends on target region)
  • Additional reagents and equipment for PCR (Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), DNA sequencing (Shendure et al., ), and Southern blotting (Brown, )
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Figures

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Literature Cited

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