%0 Journal Article
%A Kropp, Korbinian N
%A Schäufele, Tim J
%A Fatho, Martina
%A Volkmar, Michael
%A Conradi, Roland
%A Theobald, Matthias
%A Wölfel, Thomas
%A Wölfel, Catherine
%T A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.
%J PLOS ONE
%V 15
%N 9
%@ 1932-6203
%C San Francisco, California, US
%I PLOS
%M DKFZ-2020-01828
%P e0238875 -
%D 2020
%Z HI-TRON
%X 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.
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:32903281
%R 10.1371/journal.pone.0238875
%U https://inrepo02.dkfz.de/record/163037