Measurement of Mouse T Cell Receptor Excision Circles

Gregory D. Sempowski1, Maria E. Rhein1

1 Duke University, Durham, North Carolina
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.31
DOI:  10.1002/0471142735.im1031s63
Online Posting Date:  November, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit provides the protocols necessary for the quantification of mouse single joint T cell receptor excision circles (sjTRECs) generated during TCRA gene rearrangement. These nonreplicated episomal circles of DNA are generated by the recombination process used to produce antigen‐specific T cell receptors. The number of sjTRECs per mg of thymus tissue or per 100,000 lysed cells has been shown to be a molecular marker of thymopoiesis and naïve T cells. This technology is beneficial to investigators interested in quantitating the level of thymopoiesis occurring in both in vivo and in vitro mouse systems and complements traditional phenotypic analyses of thymopoiesis. The for real‐time PCR details the assay for use on a Bio‐Rad iCycler iQ. An is provided for an ABI Prism 7700. In addition, the unit includes Support Protocols for preparation of a mouse sjTREC DNA standard () and preparation of mouse thymus tissue DNA () as well as a protocol for proteinase K lysis of lymphocytes () used in the Basic and Alternate protocols.

Keywords: Mouse; Thymopoiesis; T cell receptor; Real‐time PCR

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Quantitation of Mouse TCR Delta Excision Circles by Real‐Time PCR Using the Bio‐Rad iCycleriQ Real‐Time Thermal Cycler
  • Alternate Protocol 1: Quantitation of Mouse TCR Delta Excision Circles by Real‐Time PCR Using the ABI Prism 7700 Thermal Cycler
  • Support Protocol 1: Preparation of Mouse TCR Delta sjTREC DNA Standard
  • Support Protocol 2: Preparation of Mouse Thymus Tissue DNA
  • Support Protocol 3: Proteinase K Lysis of Lymphocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Quantitation of Mouse TCR Delta Excision Circles by Real‐Time PCR Using the Bio‐Rad iCycleriQ Real‐Time Thermal Cycler

  Materials
  • DNase‐free and RNase‐free molecular‐biology grade water (Sigma)
  • Platinum Taq reaction buffer (Invitrogen)
  • 50 mM MgCl 2 (Invitrogen)
  • 10 mM dNTP (mix (see recipe)
  • 12.5 µM mouse sjTREC forward primer (see recipe)
  • 12.5 µM mouse sjTREC reverse primer (see recipe)
  • 5 µM mouse sjTREC probe (see recipe)
  • Platinum Taq DNA polymerase (Invitrogen)
  • Genomic DNA samples (1 µg/5 µl; see protocol 4) and/or proteinase K lysates of lymphocytes (50,000 cells/5 µl; see protocol 5)
  • Prediluted aliquots of 102 to 107 molecules/5 µl mouse sjTREC standards (see protocol 3)
  • PCR setup hood with UV lamp (CBS Scientific; optional)
  • 1.5‐ml polypropylene screw‐cap tubes (Sarstedt)
  • iCycler iQ 96‐well PCR plates (Bio‐Rad)
  • iCycler iQ PCR plate strip caps (Bio‐Rad)
  • Tabletop centrifuge (e.g., IEC Clinical) with swinging 96‐well plate holders
  • iCycler iQ Thermal Cycler with optical system and filter sets for detection of FAM (Bio‐Rad)

Alternate Protocol 1: Quantitation of Mouse TCR Delta Excision Circles by Real‐Time PCR Using the ABI Prism 7700 Thermal Cycler

  • Reference dye BD636 (MegaBases, Inc.; supplied as 100× stock)
  • Optical‐grade 96‐well PCR plates (Applied Biosystems)
  • Optical‐grade PCR strip caps (Applied Biosystems)
  • ABI Prism 7700 Sequence Detection System thermal cycler (Applied Biosystems)

Support Protocol 1: Preparation of Mouse TCR Delta sjTREC DNA Standard

  Materials
  • Mouse sjTREC plasmid (Gregory D. Sempowski, Duke University; gsem@duke.edu)
  • DNase‐free and RNase‐free molecular‐biology grade water (Sigma)
  • Yeast tRNA (Sigma)
  • 1.5‐ml polypropylene screw‐cap tubes (Sarstedt)
  • 15‐ml polypropylene conical tubes
  • Tabletop centrifuge (e.g., IEC Clinical)
  • Additional reagents and equipment for preparation of plasmid DNA (unit 10.3)

Support Protocol 2: Preparation of Mouse Thymus Tissue DNA

  Materials
  • Experimental mice
  • Dry ice/95% ethanol bath
  • Trizol Reagent (Invitrogen), room temperature
  • Chloroform
  • 100% and 75% molecular‐biology grade ethanol
  • 0.1 M sodium citrate in 10% ethanol (see recipe)
  • 1.8‐ml cryovials (Nunc)
  • Omni TH Homogenizer and Omni‐Tips plastic disposable Generator Probes (Omni International)
  • Ring stand
  • 5‐ml plastic scintillation vials
  • Sterile forceps
  • 55°C water bath
  • Additional reagents and equipment for euthanasia (unit 1.8) and removal of lymphoid tissues (unit 1.9) of the mouse, and spectrophotometric quantitation of DNA ( appendix 3L)
CAUTION: This protocol uses a phenol product and chloroform, and must be performed in a fume hood.

Support Protocol 3: Proteinase K Lysis of Lymphocytes

  Materials
  • 10 mM Tris⋅Cl, pH 7.8 ( appendix 2A) in DNase/RNase‐free H 2O
  • Precounted and pelleted mouse thymocytes (unit 3.1) or splenic T cell subsets (unit 3.5)
  • 19 mg/ml proteinase K (Boehringer Mannheim)
  • Eppendorf Thermomixer
  • 15 ml polypropylene centrifuge tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Chen, B.J., Cue, X.Y., Sempowski, G.D., Gooding, M.E., Liu, C.X., Haynes, B.F., and Chao, N.J. 2002. A comparison of murine T‐cell‐depleted adult bone marrow and full‐term fetal blood cells in hematopoietic engraftment and immune reconstitution. Blood 99:364‐371.
   de Villartay, J.P., Hockett, R.D., Coran, D., Korsmeyer, S.J., and Cohen, D.I. 1988. Deletion of the human T‐cell receptor delta‐gene by a site‐specific recombination: Differentiation from thymic B cell progenitors to mature B cells in vitro. Nature 335:170‐174.
   Douek, D.C., McFarland, R.D., Keiser, P.H., Gage, E.A., Massey, J.M., Haynes, B.F., Polis, M.A., Haase, A.T., Feinberg, M.B., Sullivan, J.L., Jamieson, B.D., Zack, J.A., Picker, L.J., and Koup, R.A. 1998. Changes in thymic function with age and during the treatment of HIV infection [see comments]. Nature 396:690‐695.
   Douek, D.C., Vescio, R.A., Betts, M.R., Brenchley, J.M., Hill, B.J., Zhang, L., Berenson, J.R., Collins, R.H., and Koup, R.A. 2000. Assessment of thymic output in adults after haematopoietic stem‐cell transplantation and prediction of T‐cell reconstitution. Lancet 355:1875‐1881.
   Elliott, J.F., Rock, E.P., Patten, P.A., Davis, M.M., and Chien, Y.H. 1988. The adult T‐cell receptor delta‐chain is diverse and distinct from that of fetal thymocytes. Nature 331:627‐631.
   Flores, K.G., Li, J., Sempowski, G.D., Haynes, B.F., and Hale, L.P. 1999. Analysis of the human thymic perivascular space during aging. J. Clin. Invest. 104:1031‐1039.
   Haynes, B.F., Denning, S.M., Le, P.T., and Singer, K.H. 1990. Human intrathymic T cell differentiation. Semin. Immunol. 2:67‐77.
   Hockett, R.D., Nunez, G., and Korsmeyer, S.J. 1989. Evolutionary comparison of murine and human delta T‐cell receptor deleting elements. New Biol. 1:266‐274.
   Jamieson, B.D., Douek, D.C., Killian, S., Hultin, L.E., Scripture‐Adams, D.D., Giorgi, J.V., Marelli, D., Koup, R.A., and Zack, J.A. 1999. Generation of functional thymocytes in the human adult. Immunity 10:569‐575.
   Kong, F., Chen, C.H., and Cooper, M.D. 1998. Thymic function can be accurately monitored by the level of recent T cell emigrants in the circulation. Immunity 8:97‐104.
   Min, B., McHugh, R., Sempowski, G.D., Mackall, C., Foucras, G., and Paul, W.E. 2003. Neonates support lymphopenia‐induced proliferation. Immunity 18:131‐140.
   Sempowski, G.D., Hale, L.P., Sundy, J.S., Massey, J.M., Koup, R.A., Douek, D.C., Patel, D.D., and Haynes, B.F. 2000. Leukemia inhibitory factor, oncostatin M, IL‐6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy. J. Immunol. 164:2180‐2187.
   Sempowski, G.D., Gooding, M.E., Liao, H.X., Le, P.T., and Haynes, B.F. 2002. T cell receptor excision circle assessment of thymopoiesis in aging mice. Mol. Immunol. 38:841‐848.
   Takeshita, S., Toda, M., and Yamagishi, H. 1989. Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus. EMBO J. 8:3261‐3270.
   Toda, M., Fujimoto, S., Iwasato, T., Takeshita, S., Tezuka, K., Ohbayashi, T., and Yamagishi, H. 1988. Structure of extrachromosomal circular DNAs excised from T‐cell antigen receptor alpha and delta‐chain loci. J. Mol. Biol. 202:219‐231.
Key References
   Douek et al., 2000. See above.
  Primary references for TCR excision circle assessment of thymopoiesis in humans and mice.
   Sempowski et al., 2002. See above.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library