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

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Abstract
Table of Contents
Materials
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Literature Cited

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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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)
H₂ O, 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 Xba I 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 H₂ O
Message Machine T7 Ultra kit (Ambion, cat. no. AM1345)
MegaClear kit (Ambion, cat. no. AM1908)
T₁₀ E_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 H₂ O)
Water, embryo tested (Sigma, cat. no. W1503)
T₁₀ E₁ 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)
T₁₀ E_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)F₁ 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 H₂ O 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 H₂ O 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

Figure 1. Principle of gene editing using sequence‐specific nucleases. Sequence‐specific nucleases are composed of a domain that binds to the target gene via zinc‐finger or TAL effector‐based DNA recognition motifs. The DNA binding part is fused to the nonspecific nuclease domain of Fok I. A double‐strand break is generated only by a dimer of nuclease domains each targeting one DNA strand. Two fusion proteins, one recognizing a sequence upstream of the target site and the other recognizing a downstream sequence segment, cooperate as a functional gene‐specific nuclease. The presence of a double‐strand break activates the repair mechanisms of homologous recombination (HR) and nonhomologous end ligation (NHEJ). Through HR, a preplanned genetic modification carried within a gene targeting vector is copied into the target gene. In the absence of a vector as repair template, the DSB is closed by NHEJ. This frequently leads to the loss of a variable number of nucleotides and generates knockout alleles by frameshift mutations.

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Figure 2. Gene editing in one‐cell embryos. Fertilized oocytes are collected from wild‐type mice and microinjected with mRNA for a sequence‐specific nuclease pair (Nuc1/2) that causes a double‐strand break (DSB) within the paternal or maternal copy of the target gene. (A ) Generation of knockout mice. The closure of DSBs by nonhomologous end joining (NHEJ) repair leads to the loss of nucleotides at the DSB site such that many alleles exhibit frameshift mutations, leading to a functional knockout (KO). (B ) Generation of knockin mice. Fertilized oocytes are coinjected with mRNA for a sequence‐specific nuclease (Nuc1/2) and DNA of gene targeting vector. The nuclease induces a double‐strand break (DSB) at the target site that stimulates homologous recombination (HR) of the vector with the target locus, resulting into a knockin (KI) allele. Manipulated embryos are transferred into pseudopregnant females to obtain offspring. These mice harbor heterozygous KO alleles (A) at a frequency of up to 75% or knockin alleles at a frequency to 5% (B).

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Figure 3. In vitro transcription of nuclease mRNA. Denaturing gel electrophoresis of three in vitro‐transcribed RNAs (A, B, C ) before and after polyadenylation. Control samples C ₁ taken upon the in vitro transcription () of a ZFN RNA A (A₁ , expected size: 1100 nt), a ZFN RNA B (B1, expected size: 1200 nt), and a Venus coding RNA C (C₁ , expected size: 1500 nt). The equivalent control samples C ₂ taken after RNA polyadenylation and purification exhibit a size shift of ∼500 nt, indicating successful polyadenylation. M, Millennium RNA size standard, x 1000 nucleotides.

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Figure 4. Embryo microinjection for the delivery of mRNA and targeting vector. The injection capillary is loaded with a mixture of nuclease mRNA and targeting vector DNA. (A ) In the first step of injection, a volume of the RNA/DNA mix is injected into the larger pronucleus to deliver the targeting vector to the paternal genome. (B ) During withdrawal of the injection needle, a second RNA/DNA volume is delivered into the cytoplasm for the immediate translation of nuclease mRNA.

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Figure 5. The timing of nuclease‐induced gene editing determines the genetic constitution of mutant founders. During the microinjection of nuclease mRNA, the male genome is in the process of replication. (A ) In the case where a nuclease (Nuc) processes the single copy of the target gene before replication, the genetic modification becomes transferred into all descending cells, resulting in a fully heterozygous mutant. Such founders are expected to transmit the mutant allele to 50% of their progeny. (B ) If the nuclease (Nuc) acts only on one chromatid after replication of the target gene, only half of the body cells harbor the mutation in a heterozygous state, resulting in a mosaic mutant. Mosaic founders are expected to transmit the mutant allele to 25% of their progeny.

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Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited