RRC ID |
38208
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著者 |
Arakaki A, Ooya K, Akiyama Y, Hosokawa M, Komiyama M, Iizuka A, Yamaguchi K, Matsunaga T.
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タイトル |
TCR-beta repertoire analysis of antigen-specific single T cells using a high-density microcavity array.
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ジャーナル |
Biotechnol Bioeng
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Abstract |
The antigen specificity of cytotoxic T cells, provided by T-cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single-cell level is required. In this study, we applied a high-density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR-beta gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent-labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T-cell population. The isolated single cells were then directly utilized for RT-PCR. By sequencing the amplified PCR products, antigen-specific TCR-beta repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single-cell level. This simple, accurate, and cost-effective technique for single-cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells.
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巻・号 |
106(2)
|
ページ |
311-8
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公開日 |
2010-6-1
|
DOI |
10.1002/bit.22663
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PMID |
20073087
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MeSH |
Cell Separation / instrumentation*
Equipment Design
Equipment Failure Analysis
Oligonucleotide Array Sequence Analysis / instrumentation*
Receptors, Antigen, T-Cell / genetics*
Reverse Transcriptase Polymerase Chain Reaction / instrumentation*
|
IF |
4.002
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引用数 |
10
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WOS 分野
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BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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リソース情報 |
ヒト・動物細胞 |
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