Figure 1. pcDNA3.1(+) vector with T7 promotor enabling in vitro transcription of mRNA encoding both a TCRβ and a TCRα chain.

The variable VDJβ and VJα sequences are joined in-frame with the mouse TCRβ-constant (mTRBC) and TCRα-constant (mTRAC) domains, respectively, resulting in a bicistronic open reading frame in which the TCRβ and TCRα coding sequences are separated by a self-cleaving P2A site, as described in detail by Kropp et al. [19]. mTRBC and mTRAC sequences harbor the previously described T48C and S57C mutations, respectively, resulting in an additional interchain disulfide bond [20,21]. The latter mutations in combination with the use of murine constant regions reduce mispairing of the transfected T-cell receptor (TCR) with endogenously expressed TCR chains in human T-cells. Furthermore, this allows for flow cytometric detection of cell surface expression of the gene transduced TCR by means of a monoclonal antibody against the mouse TCRβ (mTCRβ) constant domain. Downstream of this open reading frame is a 3′UTR consisting of two tandem copies of the human β-globin gene that improve stability of the resulting transcript in cells [22]. The expression cassette is embedded in the pcDNA3.1(+) vector, allowing in vitro transcription through the T7 promoter sequence.