The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen.
Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.
Hiyane
M I
MI
Centro Interdisciplinar de Terapia Gênica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
Boscardin
S B
SB
Rodrigues
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology; D005260:Female; D019672:Genes, T-Cell Receptor / Q000235:genetics* / Q000276:immunology; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006825:Hybridomas / Q000378:metabolism; D016056:Immunodominant Epitopes / Q000235:genetics* / Q000276:immunology; D007371:Interferon-gamma / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D000953:Antigens, Protozoan; D006023:Glycoproteins; D016056:Immunodominant Epitopes; D007371:Interferon-gamma; C069715:trans-sialidase; D009439:Neuraminidase