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

互联网2013-12-31

2981

Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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
Bfa l linker+	GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC
Bfa l linker−	P‐TAGTCCCTTAAGCGGAG‐AM
Nla III linker+	GTAATACGACTCACTATAGGGCTCCGCTTAAGGGACCATG
Nla III 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

^a Abbreviations: 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 (H₂ O₂ ) 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 recipe s)
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 )
Nla III (New England Biolabs)
Bfa I (New England Biolabs)
Primers (Table 11.0.8700 )
5 M NaCl
T4 DNA ligase and 10× buffer (New England Biolabs)
Bam HI (New England Biolabs)
Buffer no. 3 (New England Biolabs)
100× BSA
2× ReddyMix (Thermo Scientific)
10× buffer with 10 mM MgCl₂ (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

Figure 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.

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Figure 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.

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Figure 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.

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Figure 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.

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Figure 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.

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Figure 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.

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Videos

Literature Cited

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