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000163037 1001_ $$00000-0001-8104-7166$$aKropp, Korbinian N$$b0
000163037 245__ $$aA bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.
000163037 260__ $$aSan Francisco, California, US$$bPLOS$$c2020
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000163037 520__ $$aTo 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.
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000163037 7001_ $$aSchäufele, Tim J$$b1
000163037 7001_ $$aFatho, Martina$$b2
000163037 7001_ $$0P:(DE-He78)792fd96345efded9a78c902105479a68$$aVolkmar, Michael$$b3$$udkfz
000163037 7001_ $$aConradi, Roland$$b4
000163037 7001_ $$aTheobald, Matthias$$b5
000163037 7001_ $$aWölfel, Thomas$$b6
000163037 7001_ $$aWölfel, Catherine$$b7
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