Quantitative PCR for detection of the OT-1 transgene
<p>Abstract</p> <p>Background</p> <p>Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for th...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2005-08-01
|
| Series: | BMC Immunology |
| Online Access: | http://www.biomedcentral.com/1471-2172/6/20 |
| _version_ | 1818146080882163712 |
|---|---|
| author | Crispe Nicholas I Murray Debbie A Wright Kate O Pierce Robert H |
| author_facet | Crispe Nicholas I Murray Debbie A Wright Kate O Pierce Robert H |
| author_sort | Crispe Nicholas I |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for the SIINFEKL peptide of ovalbumin presented on k<sup>b</sup>. Although OT-1 CD8+ can be used in a variety of different experimental settings, we principally employ adoptive transfer and peptide-driven expansion of OT-1 cells in order to explore the distribution and fate of these antigen-specific OT-1 T cells. We set out to develop a quantitative PCR assay for OT-1 cells in order to assess the distribution of OT-1 CD8+ T cells in tissues that are either intrinsically difficult to dissociate for flow cytometric analysis or rendered incompatible with flow cytometric analysis through freezing or fixation.</p> <p>Results</p> <p>We show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in <it>in vivo </it>adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis.</p> <p>Conclusion</p> <p>An OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociated</p> |
| first_indexed | 2024-12-11T12:13:40Z |
| format | Article |
| id | doaj.art-26ae6fc49739403db1f93252ee8395f4 |
| institution | Directory Open Access Journal |
| issn | 1471-2172 |
| language | English |
| last_indexed | 2024-12-11T12:13:40Z |
| publishDate | 2005-08-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Immunology |
| spelling | doaj.art-26ae6fc49739403db1f93252ee8395f42022-12-22T01:07:43ZengBMCBMC Immunology1471-21722005-08-01612010.1186/1471-2172-6-20Quantitative PCR for detection of the OT-1 transgeneCrispe Nicholas IMurray Debbie AWright Kate OPierce Robert H<p>Abstract</p> <p>Background</p> <p>Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for the SIINFEKL peptide of ovalbumin presented on k<sup>b</sup>. Although OT-1 CD8+ can be used in a variety of different experimental settings, we principally employ adoptive transfer and peptide-driven expansion of OT-1 cells in order to explore the distribution and fate of these antigen-specific OT-1 T cells. We set out to develop a quantitative PCR assay for OT-1 cells in order to assess the distribution of OT-1 CD8+ T cells in tissues that are either intrinsically difficult to dissociate for flow cytometric analysis or rendered incompatible with flow cytometric analysis through freezing or fixation.</p> <p>Results</p> <p>We show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in <it>in vivo </it>adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis.</p> <p>Conclusion</p> <p>An OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociated</p>http://www.biomedcentral.com/1471-2172/6/20 |
| spellingShingle | Crispe Nicholas I Murray Debbie A Wright Kate O Pierce Robert H Quantitative PCR for detection of the OT-1 transgene BMC Immunology |
| title | Quantitative PCR for detection of the OT-1 transgene |
| title_full | Quantitative PCR for detection of the OT-1 transgene |
| title_fullStr | Quantitative PCR for detection of the OT-1 transgene |
| title_full_unstemmed | Quantitative PCR for detection of the OT-1 transgene |
| title_short | Quantitative PCR for detection of the OT-1 transgene |
| title_sort | quantitative pcr for detection of the ot 1 transgene |
| url | http://www.biomedcentral.com/1471-2172/6/20 |
| work_keys_str_mv | AT crispenicholasi quantitativepcrfordetectionoftheot1transgene AT murraydebbiea quantitativepcrfordetectionoftheot1transgene AT wrightkateo quantitativepcrfordetectionoftheot1transgene AT pierceroberth quantitativepcrfordetectionoftheot1transgene |