Lentivirus vector construction and production
DNA sequences encoding MAGE-B2-TCR alpha and beta chains separated by a
self-cleaving T2A peptide were cloned into the pALD-Lenti expression
vector (Aldevron) by isothermal assembly, with an EF1a promoter driving
TCR gene expression. A total of five MAGE-B2 TCRs were tested. DNA
sequences encoding CARs or GFP driven by a MSCV promoter were also
cloned into a lentiviral expression vector for evaluation. Lentivirus
was generated by transient transfection of HEK293 cells with third
generation LVV packaging plasmids (p-ALD-Lenti, Aldevron) and the
expression construct. Lentivirus was harvested from the supernatant 3
days post-transfection,concentrated using high-speed centrifugation,
formulated in a sucrose solution and stored at -80 degC(Gandara,
Affleck, & Stoll, 2018). Infectious titers were determined by using a
flow cytometry cell-based assay on HEK293 and Jurkat cells(Kutner,
Zhang, & Reiser, 2009).