Molecular Analysis of Mouse T Cell Receptor Expression Using PCR

Casey J. Fox1, Jayne S. Danska1

1 Hospital for Sick Children Research Institute and University of Toronto, Toronto, Canada
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.27
DOI:  10.1002/0471142735.im1027s22
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of the polymerase chain reaction (PCR) to characterize rearranged murine T cell receptor (TCR) transcripts present in primary lymphoid tissues, peripheral lymphoid tissues, and extra‐lymphoid sites of inflammation, and to quantify them either relatively or absolutely. This methodology has been used extensively to characterize TCR variability in murine thymus and liver in T cell clones, and in extralymphoid tissues where the absolute number of T cells is limited. A procedure for cloning and sequencing PCR‐amplified cDNA transcripts is provided to examine the junctional diversity of expressed genes. Also included is a method for reverse transcription of RNA into cDNA that is optimal for analysis of tissue samples where the number of T cells is limited. The detailed protocols are followed by a commentary that discusses strategies and artifacts as well as providing troubleshooting suggestions for PCR amplification and TCR analysis.

     
 
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Table of Contents

  • Basic Protocol 1: PCR Analysis of Mouse TCR Gene Expression
  • Basic Protocol 2: Estimation of the Frequency of Mouse TCR Variable Region by PCR
  • Basic Protocol 3: Quantitation of Mouse TCR Variable Region Transcripts by PCR
  • Basic Protocol 4: Sequence Analysis of the Junctional Diversity of V(D)J Regions
  • Support Protocol 1: Synthesis of cDNA for PCR Analysis of Mouse T Cell Receptor Gene Expression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: PCR Analysis of Mouse TCR Gene Expression

  Materials
  • Homogenized tissue sample(s) or single‐cell suspensions (104 to 107 cells) from normal mouse, in HBSS ( appendix 2A)
  • Sterile H 2O
  • 50 µM solutions (stored at −80°C) of primers for assessing cDNA preparations: i.e., primers for β‐actin (Table 97.80.4711) or other housekeeping gene
  • 50 µM solutions (stored at −80°C) of TCR primers (Table 97.80.4711): V‐region 5′ primers, either Vβ or Vα (specific) or VβC or VαC (degenerate), and C‐region primers, either Cβuni or Cα
  • 2 mM 4dNTP mix (e.g., Promega or unit 10.9)
  • recipe10× PCR amplification buffer (see recipe)
  • 10 U/µl Taq DNA polymerase (e.g., Perkin‐Elmer)
  • Mineral oil
  • recipe6× gel running dye (see recipe)
  • 1% to 2% agarose gel made with TAE buffer containing 0.5 µg/ml ethidium bromide (unit 10.4)
  • Alkaline hydrolysis solution: 0.5 M NaOH/1.5 M NaCl
  • Neutralization solution: 1 M Tris⋅Cl, pH 8.0 ( appendix 2A)/1.5 M NaCl
  • Absorbent paper
  • Nylon membrane: e.g., Magnacharge (Fisher) or GeneScreen Plus (Du Pont NEN)
  • 10× and 5× SSC (unit 10.6)
  • 2500 to 3500 Ci/mmol [α‐32P]CTP‐labeled, random‐primed (unit 10.10) probes for assessing cDNA preparations: e.g., for β‐actin
  • 2500 to 3500 Ci/mmol [α‐32P]CTP‐labeled, random‐primed (unit 10.10) Cα‐ and Cβ‐ or specific Vβ probes prepared from cDNA or genomic clones
  • recipePrehybridization solution (see recipe; prepare fresh)
  • recipeHybridization solution (see recipe; prepare fresh)
  • Wash solution: 0.2× SSC (unit 10.6)/0.1% SDS (filtered)
  • Thermal cycler: Perkin‐Elmer 480 or equivalent
  • UV cross‐linker (e.g., UV Stratalinker, Stratagene) or short‐wave UV source
  • Heat‐sealable plastic hybridization bags and vacuum sealer, or hybridization bottles and hybridization oven
  • Boiling water bath
  • X‐ray film and autoradiographic scanner, or PhosphorImager cassette and PhosphorImaging Scanner (Molecular Dynamics)
  • Additional reagents and equipment for PCR (unit 10.20), agarose gel electrophoresis (unit 10.4), Southern blotting by downward capillary transfer (unit 10.6), and UV cross‐linking (unit 10.6)

Basic Protocol 2: Estimation of the Frequency of Mouse TCR Variable Region by PCR

  • ImageQuant Software (Molecular Dynamics) for PhosphorImager or densitometric scanner
  • Additional reagents and equipment for amplification of TCR gene transcripts (see protocol 1)

Basic Protocol 3: Quantitation of Mouse TCR Variable Region Transcripts by PCR

  Materials
  • 0.8% agarose gel made with 1× TAE buffer containing 0.5 µg/ml ethidium bromide (unit 10.4)
  • Reagents for DNA extraction from agarose (e.g., Qiaex II Gel Extraction Kit, Qiagen, or unit 10.5)
  • Apr β‐gal+ cloning vector, either from PCR‐based cloning kit (e.g., TA Vector, Invitrogen) or conventional plasmid vector (e.g., pBluescript, from Stratagene, or pUC vector)
  • 10 µg/ml tRNA carrier in distilled H 2O
  • 100% ethanol, −20°C
  • Amps, β‐galE. coli cells (e.g., X1‐Blue, Stratagene), electrocompetent ( appendix 3N)
  • recipe2× YT medium (see recipe) containing 50 µg/ml ampicillin
  • LB agar plates containing 50 µg/ml ampicillin and treated with IPTG/Xgal ( appendix 3N)
  • 1 mg/ml sonicated E. coli DNA (Pharmacia Biotech)
  • 1 mg/ml RNase A
  • 5′Cβ–3′Cβ Rev or 5′Cα–3′Cα Rev primer
  • Electroporation apparatus (e.g., Gene Pulser, Bio‐Rad)
  • ImageQuant Software (Molecular Dynamics) for PhosphorImager or densitometric scanner
  • 37°C bacterial incubator and shaker
  • Additional reagents and equipment for RNA purification and preparation of cDNA (see protocol 5), amplification of TCR gene transcripts (see protocol 1), agarose gel electrophoresis (unit, ), extraction of DNA from agarose gel (unit 10.5), restriction enzyme digestion (unit 10.8), transformation by electroporation ( appendix 3N) or other methods (units 10.13, 10.14 & 10.16), and 10.3alkaline lysis preparation of plasmid DNA (unit 10.3).

Basic Protocol 4: Sequence Analysis of the Junctional Diversity of V(D)J Regions

  Materials
  • Homogenized tissue sample(s) or single‐cell suspensions (104 to 107 cells) from target tissue, in HBSS ( appendix 2A)
  • E. coli strain appropriate for vector and selection strategy (e.g., X1‐Blue, Stratagene)
  • LB agar plates with appropriate selective additives ( appendix 3N)
  • Phagemid vector: e.g., M13mp18, 19, 20, or 21
  • Nitrocellulose membrane: e.g., Magnacharge (Fisher)
  • [α‐32P] CTP‐labeled specific Vβ oligonucleotide probes (labeled by random priming; unit 10.10)
  • [γ‐32P]ATP‐labeled specific Jβ oligonucleotide probes (labeled using polynucleotide kinase; unit 10.10)
  • recipeOligo prehybridization solution (see recipe; prepare fresh)
  • recipeOligo hybridization solution (see recipe; prepare fresh)
  • Dideoxy sequencing reagents (United States Biochemicals)
  • Additional reagents and equipment for RNA purification and preparation of TCRβ cDNA (see protocol 5), amplification of TCR gene transcripts (see protocol 1), cloning and bacterial transformation (see protocol 3), manipulation of phagemid libraries (Ausubel et al., , and Maniatis et al., ), and sequencing using dideoxy chain termination (unit 10.26)

Support Protocol 1: Synthesis of cDNA for PCR Analysis of Mouse T Cell Receptor Gene Expression

  Materials
  • Hanks balanced salt solution (HBSS; appendix 2A)
  • Homogenized tissue sample(s) or single‐cell suspensions (104 to 107 cells) in HBSS
  • 10 µg/ml glycogen carrier (Boehringer Mannheim)
  • Guanidinium isothiocyanate solution (unit 10.11)
  • Phenol saturated with 0.5 M Tris⋅Cl, pH 8.0 (unit 10.1 use pH 4.0 Tris buffer for RNA)
  • Chloroform
  • Isopropanol, RNase free
  • 80% ethanol, RNase free, −20°C
  • DEPC‐treated sterile H 2O (unit 12.8)
  • 20 pg/µl oligo‐dT primer (Promega)
  • recipe5× RT reaction buffer (see recipe)
  • 5 mM 4dNTP mix (Promega or unit 10.9)
  • 1 mg/ml molecular‐grade bovine serum albumin (BSA; Boehringer Mannheim)
  • Avian myoblastosis virus (AMV) reverse transcriptase (RTase; Promega; dilute required quantity in DEPC‐treated water to obtain 3 U/µl concentration)
  • RNase inhibitor: 33 U/µl RNasin (Promega)
  • 1 mg/ml sonicated E. coli DNA (Pharmacia Biotech)
  • 1.5‐ml microcentrifuge tubes, sterile
NOTE: All solutions should be prepared with sterile water treated with diethylpyrocarbonate (DEPC; unit 12.8) to remove contaminating RNase activity.
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Figures

Videos

Literature Cited

Literature Cited
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