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-β 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-β 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.
Original language | English |
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Pages (from-to) | 311-318 |
Number of pages | 8 |
Journal | Biotechnology and Bioengineering |
Volume | 106 |
Issue number | 2 |
DOIs | |
Publication status | Published - 2010 Jun 1 |
Externally published | Yes |
Keywords
- Gene expression analysis
- Microarray analytical devices
- Single-cell
- T-cell antigen receptor specificity
- T-cell receptor beta genes
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology