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@ARTICLE{Kropp:163037,
      author       = {K. N. Kropp and T. J. Schäufele and M. Fatho and M.
                      Volkmar$^*$ and R. Conradi and M. Theobald and T. Wölfel
                      and C. Wölfel},
      title        = {{A} bicistronic vector backbone for rapid seamless cloning
                      and chimerization of αβ{T}-cell receptor sequences.},
      journal      = {PLOS ONE},
      volume       = {15},
      number       = {9},
      issn         = {1932-6203},
      address      = {San Francisco, California, US},
      publisher    = {PLOS},
      reportid     = {DKFZ-2020-01828},
      pages        = {e0238875 -},
      year         = {2020},
      note         = {HI-TRON},
      abstract     = {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.},
      cin          = {FM01 / D200 / D190},
      ddc          = {610},
      cid          = {I:(DE-He78)FM01-20160331 / I:(DE-He78)D200-20160331 /
                      I:(DE-He78)D190-20160331},
      pnm          = {314 - Tumor immunology (POF3-314)},
      pid          = {G:(DE-HGF)POF3-314},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:32903281},
      doi          = {10.1371/journal.pone.0238875},
      url          = {https://inrepo02.dkfz.de/record/163037},
}