Codon-optimized rhesus-derived iNKT TCR Cloning

Codon-optimized rhesus-derived iNKT cell TCR sequences were assembled into a TCR cassette (Linnemann et al. 2013 (link)). In this construct, the TCR-α and TCR-β chain constant regions are replaced with modified murine TCR constant regions to facilitate measurement of TCR expression and to encourage pairing between exogenous TCR chains. The TCR cassettes were synthesized through Thermo Fisher GeneArt Synthesis service and were then cloned into pRRL.PPT.MP.GFPpre (Jing et al. 2016 (link); Zhou et al. 2012 (link)) using BamHI and SalI restriction enzymes (New England BioLabs, Ipswich, MA). All plasmids were purified using Maxi Prep kits (Qiagen, Hilden, Germany).