A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.
To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ com...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2020-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0238875 |
| _version_ | 1818588723095273472 |
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| author | Korbinian N Kropp Tim J Schäufele Martina Fatho Michael Volkmar Roland Conradi Matthias Theobald Thomas Wölfel Catherine Wölfel |
| author_facet | Korbinian N Kropp Tim J Schäufele Martina Fatho Michael Volkmar Roland Conradi Matthias Theobald Thomas Wölfel Catherine Wölfel |
| author_sort | Korbinian N Kropp |
| collection | DOAJ |
| description | To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRβ V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αβTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order β-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells. |
| first_indexed | 2024-12-16T09:29:17Z |
| format | Article |
| id | doaj.art-945f4e5a0b6a43829474d7d811db6425 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-16T09:29:17Z |
| publishDate | 2020-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-945f4e5a0b6a43829474d7d811db64252022-12-21T22:36:35ZengPublic Library of Science (PLoS)PLoS ONE1932-62032020-01-01159e023887510.1371/journal.pone.0238875A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.Korbinian N KroppTim J SchäufeleMartina FathoMichael VolkmarRoland ConradiMatthias TheobaldThomas WölfelCatherine WölfelTo facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRβ V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αβTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order β-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.https://doi.org/10.1371/journal.pone.0238875 |
| spellingShingle | Korbinian N Kropp Tim J Schäufele Martina Fatho Michael Volkmar Roland Conradi Matthias Theobald Thomas Wölfel Catherine Wölfel A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences. PLoS ONE |
| title | A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences. |
| title_full | A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences. |
| title_fullStr | A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences. |
| title_full_unstemmed | A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences. |
| title_short | A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences. |
| title_sort | bicistronic vector backbone for rapid seamless cloning and chimerization of αβt cell receptor sequences |
| url | https://doi.org/10.1371/journal.pone.0238875 |
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