Generation of Spatio‐Temporally Controlled Targeted Somatic Mutations in the Mouse

Daniel Metzger1, Pierre Chambon1

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, and Collège de France, Illkirch, France
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo100128
Online Posting Date:  March, 2011
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Abstract

The generation of ligand-activated site-specific Cre recombinases has led to the development of cell type–specific temporally controlled targeted somatic mutagenesis in the mouse. We illustrate this technique using K14-Cre-ERT2 transgenic mice that express the tamoxifen (tam)-activatable Cre-ERT2 recombinase in epidermal basal keratinocytes to induce mutations in epidermal keratinocytes of adult mice. Our highly reproducible technique, based on induction of Cre-ERT2 recombinase activity by tamoxifen administration at low doses (once daily 100-µg intraperitoneal injection for 5 days), has allowed the generation of site-directed somatic mutations of numerous genes in mouse epidermal keratinocytes, and several mouse models of human diseases. The present step-by-step protocol describes how to introduce temporally controlled targeted mutations in epidermal keratinocytes of adult mice. Curr. Protoc. Mouse Biol. 1:55-70. © 2011 by John Wiley & Sons, Inc.

Keywords: Cre-ERT2; tamoxifen; loxP; keratinocytes; skin

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Generation of Temporally Controlled Mutations in Skin Using Mice Expressing Cre-ERT2 Selectively in Keratinocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Generation of Temporally Controlled Mutations in Skin Using Mice Expressing Cre-ERT2 Selectively in Keratinocytes

 Materials
  • K14-Cre-ERT2 mice (Indra et al., 2000; Li et al., 2000; US patent no.7112715 and European patent no.1 692 936 cover commercial use of Cre-ERT2 expressing mice)
  • GeneXL2/+ or GeneXL2/L2 mice, which bear one or two floxed target alleles
  • Custom-designed oligo primers (see Table 1 and Figs. 1 and 2; also see recipe)
  • Direct PCR lysis solution (see recipe)
  • Cre PCR master mix (see Table 2)
  • GeneX PCR master mix (see Table 3)
  • Ethidium bromide–stained 2.0% agarose gel in TBE electrophoresis buffer (see recipe) (Armstrong and Schulz, 2008)
  • DNA Ladder Gene Ruler (Euromedex, cat. no. SM0331)
  • Tamoxifen ( see recipe)
  • Isoflurane
  • 70% (v/v) ethanol
  • Dispase solution (see recipe)
  • Proteinase K digestion buffer (see recipe)
  • Ethanol (EtOH)
  • Sterile water
  • Tris×Cl, pH 8.0
  • 1:1 phenol:chloroform (see recipe)
  • 1.5-ml microcentrifuge tubes
  • 55°C incubator
  • 85°C water bath
  • 0.2-ml PCR microtubes (Dominique Dutscher, cat. no. 01600)
  • Thermal cycler (Gene Amp PCR 9700; Applied Biosystems)
  • Gloves (Laboratories Euromedis, cat. no. 127587)
  • Syringe (1-ml equipped with a 25-G needle; Terumo, cat. no. BS-01 H2516)
  • Gas anesthesia station for rodents (TEM)
  • Animal electric shaver
  • Surgical instruments: dissection scissors, straight surgical forceps, needle holder
  • Suture materials (Ethibond Excel polyester 3-0; Ethicon, cat. no. X32040)
  • Microcentrifuge (Eppendorf, cat. no. 5415D)
  • Additional reagents and equipment for running PCR products on an ethidium bromide–stained 2.0% agarose gel in TBE electrophoresis buffer (Armstrong and Schulz, 2008)

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.
 FigureFigure 1. Schematic structure of the K14-Cre-ERT2 transgene. The human K14 promoter, the Cre-ERT2 coding sequence, and the simian virus 40 polyadenylation signal (polyA) are represented by blue, yellow, and purple boxes, respectively. The rabbit -globin intron and splice donor- and acceptor-sites are depicted by a line and pink boxes, respectively. The position of the PCR primers TK139 and TK141, and the length of the PCR-amplified DNA segment are indicated. The sequence of the K14-Cre-ERT2 transgene is available upon request.
 FigureFigure 2. Schematic diagram of a target GeneX WT allele, a floxed GeneX L2 allele, and a GeneX L- allele generated by Cre-mediated excision. Two exons (En and En+1) of GeneX are boxed. P1, P2, and P3 are PCR primers used to identify GeneX WT, L2, and L- alleles are indicated. Arrowheads represent loxP sites.
 
Table 1. Custom Designed Oligonucleotides
For Cre-ERT2 (see Fig. 1)
TK1395¢-ATTTGCCTGCATTACCGGTC-3¢
TK 1415¢-ATCAACGTTTTGTTTTCGGA-3¢
For internal control (myogenin)
ADV285¢-TTACGTCCATCGTGGACAGC-3¢
ADV305¢-TGGGCTGGGTGTTAGCCTTA-3¢
For GeneX WT, L2 and L- alleles, P1, P2, and P3
See Figure 2
For RXR alleles
P1, BAA239            5¢-TCAAGTGAGGTGGACATTAGGATG-3¢
P2, BAA982            5¢-CTGGAAGAGGATGGGCACTATTCT-3¢
P3, BAA983            5¢-AAACTGCAAGTGGCCTTGAGAAGAA-3¢
 
Table 2. Cre PCR Master Mix for 10 Reactions to Identify the K14-Cre-ERT2 Transgene
Volume of reagent
ReagentInitial concentration of reagentFinal concentration of reagent1 reaction    10 reactions
PCR buffer (see recipe)10×1×3 µl30 µl
dNTPs (see recipe)10 mM each100 µM0.3 µl3 µl
Forward primer TK139100 µM0.33 µM0.1 µl1 µl
Reverse primer TK141100 µM0.33 µM0.1 µl1 µl
Forward primer ADV28100 µM0.33 µM0.1 µl1 µl
Reverse primer ADV30100 µM0.33 µM0.1 µl1 µl
Taq DNA polymerasea5 u/µl1 U0.2 µl2 µl
Water24.1 µl241 µl
 a5 U/µl; Sigma, cat. no. D4545.
 
Table 3. GeneX PCR Master Mix for 10 Reactions to Identify GeneX WT and L2 Alleles
Volume of reagent
ReagentInitial concentration of reagentFinal concentration of reagent1 reaction10 reactions
PCR buffer (see recipe)10×1×3 µl30 µl
dNTPs (see recipe)10 mM each100 µM0.3 µl3 µl
Forward primer P1 (i.e., BAA239 for RXR)100 µM0.33 µM0.1 µl1 µl
Reverse primer P2 (i.e., BAA982 for RXR)100 µM0.33 µM0.1 µl1 µl
Taq polymerasea5 u/µl1 U0.2 µl2 µl
Water24.3 µl243 µl
 a5 U/µl; Sigma, cat. no. D4545.
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Figures

  •  FigureFigure 1. Schematic structure of the K14-Cre-ERT2 transgene. The human K14 promoter, the Cre-ERT2 coding sequence, and the simian virus 40 polyadenylation signal (polyA) are represented by blue, yellow, and purple boxes, respectively. The rabbit -globin intron and splice donor- and acceptor-sites are depicted by a line and pink boxes, respectively. The position of the PCR primers TK139 and TK141, and the length of the PCR-amplified DNA segment are indicated. The sequence of the K14-Cre-ERT2 transgene is available upon request.
    View Image
  •  FigureFigure 2. Schematic diagram of a target GeneX WT allele, a floxed GeneX L2 allele, and a GeneX L- allele generated by Cre-mediated excision. Two exons (En and En+1) of GeneX are boxed. P1, P2, and P3 are PCR primers used to identify GeneX WT, L2, and L- alleles are indicated. Arrowheads represent loxP sites.
    View Image
  •  FigureFigure 3. Identification of K14-Cre-ERT2 transgenic mice by PCR-mediated tail genomic DNA amplification. PCR-amplified DNA segments were run on a 2% agarose gel. Lanes 5 to 9, amplification products from 5 pups (Pu 1 to 5). Lanes 2 and 3, amplification products from K14-Cre-ERT2 and WT mice, respectively. Lane 4, PCR reaction without genomic DNA (lysis solution). Lane 1, DNA ladder (L). The size of the DNA segments is given in base pairs. Cre, DNA segment amplified from the K14-Cre-ERT2 transgene with the primer pair TK139 and TK141 (Fig. 1). IC (Internal control): DNA segment amplified from an endogenous mouse gene (myogenin) with the primer pair ADV28 and ADV30. Pu1, Pu3, and Pu4 are transgenic for K14-Cre-ERT2; Pu2 and Pu5 are not.
    View Image
  •  FigureFigure 4. Identification of RXR WT and L2 alleles by PCR-mediated tail genomic DNA amplification. PCR-amplified DNA segments with the primer BAA239 (P1) and BAA982 (P2) were run on a 2% agarose gel. Lanes 5 to 9, amplification products from 5 pups (Pu 1 to 5). Lanes 2 and 3, amplification products from floxed RXRL2/L2 and WT mice, respectively. Lane 4, PCR reaction without genomic DNA (lysis solution). Lane 1, DNA ladder (L). The size of the DNA segments is in base pairs. The position of the PCR product amplified with BAA239 and BAA982 from RXR WT (WT; 158 bp) and L2 floxed alleles (L2; 199 bp) are indicated. Pu1 and Pu5 show RXR WT alleles; Pu2, Pu3, and Pu4 show one RXR WT allele and one RXR L2 allele.
    View Image
  •  FigureFigure 5. Analysis of K14-Cre-ERT2 -mediated recombination of “floxed” RXR in genomic DNA of adult skin. L2 and L- RXR alleles were identified by PCR analysis of genomic DNA extracted from epidermis “E” or dermis “D” isolated from the tail two weeks after administration of either tamoxifen (tam) or vehicle (veh) to K14-Cre-ER T2(tg/0)/RXR L2/L2 and K14-Cre-ER T2(0/0) /RXRL2/L2 mice (lanes 5 to 10), as indicated. Lanes 2 and 3, amplification products from RXRL2/L2 and RXRL-/+ mice, respectively. The PCR products corresponding to the RXR WT (158 bp) and RXR L2 (199 bp) alleles were amplified with primers BAA239 (P1) and BAA982 (P2) (top panel); the PCR products corresponding to RXR L- alleles (133 bp) were amplified with primers BAA239 (P1) and BAA983 (P3) (bottom panel). Lane 1, DNA ladder (L). The size of the DNA segments is given in base pairs.
    View Image

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