A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.

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, a

SUBMITTER: Kropp KN 

PROVIDER: S-EPMC7480877 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature