A Comprehensive Guide to Sleeping Beauty–Based Somatic Transposon Mutagenesis in the Mouse

Branden Moriarity1, David A. Largaespada1

1 Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110087
Online Posting Date:  September, 2011
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Abstract

Recent advances in whole genome analyses made possible by next‐generation DNA sequencing, high‐density array comparative genome hybridization (aCGH), and other technologies have made it apparent that cancers harbor numerous genomic changes. However, without functional correlation or validation, it has proven difficult to determine which genetic changes are necessary or sufficient to produce cancer. Thus, it is still necessary to perform unbiased functional studies using model organisms to help interpret the results of whole genome analyses of human tumors. To this end, a Sleeping Beauty (SB) transposon–based mutagenesis technology was developed to identify genes that, when mutated, can cause cancer. Herein a detailed methodology to initiate and carry out an SB transposon mutagenesis screen is described. Although this system might be used to identify genes involved with many cellular phenotypes, it has been primarily implemented for cancer. Thus, SB transposon somatic cell screens for cancer development are highlighted. Curr. Protoc. Mouse Biol. 1:347‐368 © 2011 by John Wiley & Sons, Inc.

Keywords: Sleeping Beauty; cancer; mutagenesis screen; transposon; mouse

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Breeding and Genotyping a Cohort of Mice Undergoing Somatic Transposon Mutagenesis
  • Basic Protocol 2: Verifying Transposase Expression and Transposon Mobilization
  • Alternate Protocol 1: Transposon PCR Excision
  • Basic Protocol 3: Identification of Transposon Insertion Sites by LM‐PCR and High‐Throughput Sequencing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Breeding and Genotyping a Cohort of Mice Undergoing Somatic Transposon Mutagenesis

  Materials
  • Breeding pairs of all necessary transgenic mice (8‐ to 20‐weeks of age)
    • R26‐LSL‐SB11 (Dupuy et al., )
    • T2/Onc Concatemer (Collier et al., ; Dupuy et al., )
    • TSP‐Cre recombinase (http://nagy.mshri.on.ca/cre_new/index.php)
    • Conditional or non‐conditional predisposing transgene (optional)
  • IACUC‐approved food and water
  • SDS lysis buffer (see recipe)
  • Proteinase K (see recipe)
  • Phenol (Sigma‐Aldrich)
  • Chloroform (Sigma‐Aldrich)
  • Isopropanol, ice cold
  • 70% ethanol
  • TE buffer (see recipe)
  • 1.1× ReddyMix (Thermo Scientific)
  • Primers (Table 11.0.8700)
  • Genomic DNA (negative and positive controls)
  • 1% agarose gel in 1× TAE buffer
  • 1× TAE (from 50× stock) (see recipe)
  • 100‐bp Quanti‐Marker (BioExpress)
  • Ethidium bromide (10 mg/ml) (Sigma‐Aldrich)
  • IACUC‐approved animal housing facility
  • IACUC‐approved animal cages and bedding
  • Sterile 1.5‐ml microcentrifuge tubes
  • 55°C shaking incubator
  • Microcentrifuge, room temperature and 4°C
  • Spectrophotometer
  • 0.5‐ml thin‐walled PCR tubes
  • Thermal cycler
  • Gel electrophoresis apparatus and power source
  • UV light box
  • Gel photography equipment
    Table 1.0.1   MaterialsPrimer List

    Primer Sequence 5′‐3′ a
    R26‐LSL‐WT reverse CCCCAGATGACTACCTATCCTCCC
    R26‐LSL‐WT forward CTGTTTTGGAGGCAGGAA
    R26‐LSL‐SB11 reverse CTAAAAGGCCTATCACAAAC
    T2/Onc forward CGCTTCTCGCTTCTGTTCGC
    T2/Onc reverse CCACCCCCAGCATTCTAGTT
    Excision assay forward TGTGCTGCAAGGCGATTA
    Excision assay reverse ACCATGATTACGCCAAGC
    Generic Cre forward TTCGGCTATACGTAACAGGG
    Generic Cre reverse TCGATGCAACGAGTGATGAG
    Bfal linker+ GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC
    Bfal linker− P‐TAGTCCCTTAAGCGGAG‐AM
    NlaIII linker+ GTAATACGACTCACTATAGGGCTCCGCTTAAGGGACCATG
    NlaIII linker− P‐GTCCCTTAAGCGGAGCC‐AM
    Primary Splink IRDR right GCTTGTGGAAGGCTACTCGAAATGTTTGACCC
    Primary Splink IRDR left CTGGAATTTTCCAAGCTGTTTAAAGGCACAGTCAAC
    Primary Splink linker GTAATACGACTCACTATAGGGC
    Secondary Splink IRDR right (Illumina) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT(N)10 AAGTGTATGTAAACTTCCGACTTCAA
    Secondary Splink IRDR left (Illumina) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT(N)10 AGGTGTATGTAAACTTCCGACTTCAA
    Secondary linker (Illumina) CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTAGGGCTCCGCTTAAGGGAC

     aAbbreviations: P, phosphorylation; AM, amino modifier.

Basic Protocol 2: Verifying Transposase Expression and Transposon Mobilization

  Materials
  • Prepared slides of tissue of interest cut from paraffin blocks
  • Citrosolv (Fisher Scientific)
  • Ethanol (Sigma‐Aldrich)
  • Phosphate buffered saline, pH 7.5 (Fisher Scientific)
  • Unmasking solution (Vector Laboratories)
  • 3% hydrogen peroxide (H 2O 2) diluted in water (Sigma‐Aldrich)
  • M.O.M. mouse Ig blocking reagent (Vector Laboratories)
  • 1× PBST (1× PBS with 0.1% Tween 20)
  • Anti‐SB11 antibody (mouse monoclonal clone 324622) (R&D Systems)
  • VectaStain Elite ABC reagent (Vector Laboratories)
  • Diaminobenzidene (DAB) substrate kit (Vector Laboratories)
  • Harris hematoxylin (Fisher Scientific)
  • Permount (Fisher Scientific)
  • 1‐liter beakers
  • Microwave
  • ImmunoEdge Pen (Vector Laboratories)
  • Humidity chamber (see recipes)
  • 24 × 50 no. 1.5 coverslips (Thermo Scientific)
  • Microscope

Alternate Protocol 1: Transposon PCR Excision

  Materials
  • 2× ReddyMix (Thermo Scientific)
  • Primers (Table 11.0.8700)
  • DNA samples
  • DNase/RNase‐free water (Qiagen)
  • Genomic DNA (negative and positive controls)
  • 1% agarose gel in 1× TAE buffer
  • 1× TAE (from 50× stock; see recipe)
  • 100‐bp Quanti‐marker (BioExpress)
  • Ethidium bromide (10 mg/ml) (Sigma‐Aldrich)
  • 0.5‐ml thin‐walled PCR tubes
  • Thermal cycler
  • Gel electrophoresis apparatus and power source
  • UV light box
  • Gel photography equipment
  • Additional reagents and equipment for DNA extraction (see protocol 1)

Basic Protocol 3: Identification of Transposon Insertion Sites by LM‐PCR and High‐Throughput Sequencing

  Materials
  • Genomic DNA (see protocol 1)
  • TE buffer (see recipe)
  • NlaIII (New England Biolabs)
  • BfaI (New England Biolabs)
  • Primers (Table 11.0.8700)
  • 5 M NaCl
  • T4 DNA ligase and 10× buffer (New England Biolabs)
  • BamHI (New England Biolabs)
  • Buffer no. 3 (New England Biolabs)
  • 100× BSA
  • 2× ReddyMix (Thermo Scientific)
  • 10× buffer with 10 mM MgCl 2 (Roche Scientific)
  • 25 mM dNTPs (Roche Scientific)
  • Fast‐Start Taq polymerase (Roche Scientific)
  • 2% agarose gel in 1× TAE buffer
  • 1× TAE (from 50× stock; see recipe)
  • 6× DNA loading buffer (see recipe)
  • Ethidium bromide (10 mg/ml) (Sigma‐Aldrich)
  • 96‐well PCR plates
  • 37°C incubator
  • 80° and 95°C heating blocks
  • Qiagen MinElute 96 UF plates (Qiagen)
  • Orbital shaker
  • Gel electrophoresis apparatus and power source
  • UV light box
  • Gel photography equipment
  • Spectrophotometer
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for DNA isolation (see protocol 1)
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Figures

  •   FigureFigure 1. Outline of crosses necessary to generate experimental class animals undergoing transposon mutagenesis. (A,B) Experimental class animals for screens utilizing no predisposing background or a non‐conditional background are generated by breeding animals homozygous for both R26‐LSL‐SB11 and the T2/Onc concatemer to animals that are heterozygous for the Cre (TSP‐Cre) of interest or homozygous for the non‐conditional predisposing background and heterozygous for the TSP‐Cre, respectively. (C) Experimental class animals for screens utilizing a conditional predisposing background are generated by breeding animals homozygous for both R26‐LSL‐SB11 and the conditional predisposing background to mice homozygous for the T2/Onc concatemer and heterozygous for the TSP‐Cre of interest.
  •   FigureFigure 2. An example of PCR genotyping results for Cre recombinase, T2/Onc, and R26‐LSL‐SB11. Cre genotyping primers should produce a product of 482 bp. The R26‐LSL‐SB11 genotyping PCR utilizes a three‐primer PCR to amplify both the wild‐type (WT) R26 and the knock‐ in R26‐LSL‐SB11 alleles in a single reaction with wild type producing a 420‐bp product and the SB11 knock‐in producing a 266‐bp product. T2/Onc genotyping primers should produce a product of 264 bp.
  •   FigureFigure 3. Shown are example photomicrographs of immunohistochemical results for the SB transposase protein counterstained with hematoxylin. Staining of tumor cells expressing SB show robust brown horseradish peroxidase staining that is most pronounced in the nucleus where the protein is localized. Negative control staining lacking primary antibody should be devoid of brown horseradish peroxidase staining but still show counterstaining with hematoxylin.
  •   FigureFigure 4. An example of a PCR excision assay results from a panel of transposon mutagenesis induced tumors. Tumors positive for transposon mobilization should produce a 225‐bp product. If no transposition has occurred, then a 2.2‐kb product should be observed, as shown for tumor 2, which was negative for the SB transposase and developed as a background tumor in this experiment. Some tumors may be composed of a mix of cells positive and negative for transposition and will thus produce both the 225‐bp and 2.2‐kb products, as shown in tumor 5 and 6.
  •   FigureFigure 5. Flowchart outlining the molecular details at each step of the ligation‐mediated PCR (LM‐PCR) procedure leading to the final products that contain all the necessary elements for high‐throughput Illumina sequencing to identify the genomic site of transposon integrations.
  •   FigureFigure 6. An example of ligation‐mediated PCR (LM‐PCR) results from a panel of tumors induced by transposon mutagenesis. LM‐PCR products should appear as smears as they contain many different sized products that correspond to many different amplified transposon‐genomic DNA junction products. Wild‐type mouse DNA and water should not produce PCR products of any kind.

Videos

Literature Cited

Literature Cited
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