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 1: 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 1: Generation of Temporally Controlled Mutations in Skin Using Mice Expressing Cre‐ERT2 Selectively in Keratinocytes

  Materials
  • K14‐Cre‐ERT2 mice (Indra et al., ; Li et al., ; 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 10.1.2800 and Figs. and ; also see recipe)
  • Direct PCR lysis solution (see recipe)
  • Cre PCR master mix (see Table 10.1.2800)
  • GeneX PCR master mix (see Table 10.1.2800)
  • Ethidium bromide–stained 2.0% agarose gel in TBE electrophoresis buffer (see recipe) (Armstrong and Schulz, )
  • 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, )
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.
Table 0.1.1   MaterialsCustom Designed OligonucleotidesCre PCR Master Mix for 10 Reactions to Identify the K14‐Cre‐ERT2 TransgeneGeneX PCR Master Mix for 10 Reactions to Identify GeneX WT and L2 Alleles

For Cre‐ERT2 (see Fig. 1)
TK139 5′‐ATTTGCCTGCATTACCGGTC‐3′
TK 141 5′‐ATCAACGTTTTGTTTTCGGA‐3′
For internal control (myogenin)
ADV28 5′‐TTACGTCCATCGTGGACAGC‐3′
ADV30 5′‐TGGGCTGGGTGTTAGCCTTA‐3′
For GeneX WT, L2 and L‐ alleles, P1, P2, and P3
See Figure
For RXRα alleles
P1, BAA239    5′‐TCAAGTGAGGTGGACATTAGGATG‐3′
P2, BAA982    5′‐CTGGAAGAGGATGGGCACTATTCT‐3′
P3, BAA983    5′‐AAACTGCAAGTGGCCTTGAGAAGAA‐3′
Volume of reagent
Reagent Initial concentration of reagent Final concentration of reagent 1 reaction  10 reactions
PCR buffer (see recipe) 10× 3 µl 30 µl
dNTPs (see recipe) 10 mM each 100 µM 0.3 µl 3 µl
Forward primer TK139 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer TK141 100 µM 0.33 µM 0.1 µl 1 µl
Forward primer ADV28 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer ADV30 100 µM 0.33 µM 0.1 µl 1 µl
Taq DNA polymerase a 5 u/µl 1 U 0.2 µl 2 µl
Water 24.1 µl 241 µl
Volume of reagent
Reagent Initial concentration of reagent Final concentration of reagent 1 reaction 10 reactions
PCR buffer (see recipe) 10× 3 µl 30 µl
dNTPs (see recipe) 10 mM each 100 µM 0.3 µl 3 µl
Forward primer P1 (i.e., BAA239 for RXRα) 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer P2 (i.e., BAA982 for RXRα) 100 µM 0.33 µM 0.1 µl 1 µl
Taq polymerase b 5 u/µl 1 U 0.2 µl 2 µl
Water 24.3 µl 243 µl

Table 0.1.2   MaterialsCustom Designed OligonucleotidesCre PCR Master Mix for 10 Reactions to Identify the K14‐Cre‐ERT2 TransgeneGeneX PCR Master Mix for 10 Reactions to Identify GeneX WT and L2 Alleles

For Cre‐ERT2 (see Fig. 1)
TK139 5′‐ATTTGCCTGCATTACCGGTC‐3′
TK 141 5′‐ATCAACGTTTTGTTTTCGGA‐3′
For internal control (myogenin)
ADV28 5′‐TTACGTCCATCGTGGACAGC‐3′
ADV30 5′‐TGGGCTGGGTGTTAGCCTTA‐3′
For GeneX WT, L2 and L‐ alleles, P1, P2, and P3
See Figure
For RXRα alleles
P1, BAA239    5′‐TCAAGTGAGGTGGACATTAGGATG‐3′
P2, BAA982    5′‐CTGGAAGAGGATGGGCACTATTCT‐3′
P3, BAA983    5′‐AAACTGCAAGTGGCCTTGAGAAGAA‐3′
Volume of reagent
Reagent Initial concentration of reagent Final concentration of reagent 1 reaction  10 reactions
PCR buffer (see recipe) 10× 3 µl 30 µl
dNTPs (see recipe) 10 mM each 100 µM 0.3 µl 3 µl
Forward primer TK139 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer TK141 100 µM 0.33 µM 0.1 µl 1 µl
Forward primer ADV28 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer ADV30 100 µM 0.33 µM 0.1 µl 1 µl
Taq DNA polymerase a 5 u/µl 1 U 0.2 µl 2 µl
Water 24.1 µl 241 µl
Volume of reagent
Reagent Initial concentration of reagent Final concentration of reagent 1 reaction 10 reactions
PCR buffer (see recipe) 10× 3 µl 30 µl
dNTPs (see recipe) 10 mM each 100 µM 0.3 µl 3 µl
Forward primer P1 (i.e., BAA239 for RXRα) 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer P2 (i.e., BAA982 for RXRα) 100 µM 0.33 µM 0.1 µl 1 µl
Taq polymerase b 5 u/µl 1 U 0.2 µl 2 µl
Water 24.3 µl 243 µl

 a5 U/µl; Sigma, cat. no. D4545.
Table 0.1.3   MaterialsCustom Designed OligonucleotidesCre PCR Master Mix for 10 Reactions to Identify the K14‐Cre‐ERT2 TransgeneGeneX PCR Master Mix for 10 Reactions to Identify GeneX WT and L2 Alleles

For Cre‐ERT2 (see Fig. 1)
TK139 5′‐ATTTGCCTGCATTACCGGTC‐3′
TK 141 5′‐ATCAACGTTTTGTTTTCGGA‐3′
For internal control (myogenin)
ADV28 5′‐TTACGTCCATCGTGGACAGC‐3′
ADV30 5′‐TGGGCTGGGTGTTAGCCTTA‐3′
For GeneX WT, L2 and L‐ alleles, P1, P2, and P3
See Figure
For RXRα alleles
P1, BAA239    5′‐TCAAGTGAGGTGGACATTAGGATG‐3′
P2, BAA982    5′‐CTGGAAGAGGATGGGCACTATTCT‐3′
P3, BAA983    5′‐AAACTGCAAGTGGCCTTGAGAAGAA‐3′
Volume of reagent
Reagent Initial concentration of reagent Final concentration of reagent 1 reaction  10 reactions
PCR buffer (see recipe) 10× 3 µl 30 µl
dNTPs (see recipe) 10 mM each 100 µM 0.3 µl 3 µl
Forward primer TK139 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer TK141 100 µM 0.33 µM 0.1 µl 1 µl
Forward primer ADV28 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer ADV30 100 µM 0.33 µM 0.1 µl 1 µl
Taq DNA polymerase a 5 u/µl 1 U 0.2 µl 2 µl
Water 24.1 µl 241 µl
Volume of reagent
Reagent Initial concentration of reagent Final concentration of reagent 1 reaction 10 reactions
PCR buffer (see recipe) 10× 3 µl 30 µl
dNTPs (see recipe) 10 mM each 100 µM 0.3 µl 3 µl
Forward primer P1 (i.e., BAA239 for RXRα) 100 µM 0.33 µM 0.1 µl 1 µl
Reverse primer P2 (i.e., BAA982 for RXRα) 100 µM 0.33 µM 0.1 µl 1 µl
Taq polymerase b 5 u/µl 1 U 0.2 µl 2 µl
Water 24.3 µl 243 µl

 b5 U/µl; Sigma, cat. no. D4545.
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Figures

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Literature Cited

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