2.1. Plasmid construction and lentivirus preparation
The target TR1419CAR consisted of
TR1419 scFv, CD28 or 4-1BB costimulatory domains, and
CD3-ζ signaling domain [24,25]. They
were obtained by overlap PCR amplification, PCR products were clone into
lentivirus vector pWPXL and verified by enzymatic digestion and
sequencing. To produce the lentivirus supernatant,
8×105 HEK-293T cells were plated in 6-well. After 24h,
293T cells were transfected with pMD.2G encoding VSV-G envelope, pSPAX2
lentivirus plasmid, and the CAR-pWPXL plasmids by using xfect
Transfection Reagent (Takara), according to the manufacturer’s
instructions. The lentivirus supernatants were collected and filtered
with 0.45 μM filter at 48h after transfection.