JP-2021507713-A5 -

Dates

Publication Date

20230523

Application Date

20181219

Description

Cell Culture and Co-transfection 293T cells were seeded in 6-well plates containing complete DMEM with 10% FBS, penicillin, and streptomycin. Using PEI (1.7 μg each plasmid), the cells were co-transfected with equal amounts of the VEEV RSVp-NLuc construct containing nsp1-4 (VR) (SEQ ID NO: 5) or lacking the nsp1-4 fragment (BB), along with a helper plasmid encoding the capsid and a helper plasmid encoding the glycoprotein E1-6K-E2-E3. The helper plasmid combinations were (SEQ ID NOs: 1 and 3), (SEQ ID NOs: 1 and 4), (SEQ ID NOs: 2 and 3), or (SEQ ID NOs: 2 and 4). The cells were incubated with the transfection mixture at 37°C for approximately 3 hours, after which the transfection mixture was removed, the cells were washed with 1×PBS, and fresh DMEM was added. Packaged viral replicon particles (VRPs) were recovered at either 48 hpt (time after transfection) or 72 hpt. To recover the VRPs, the culture supernatant of the transfected cells was obtained by centrifuging the cell culture at 1200 rpm for 5 minutes at 4°C to pellet all cell debris. The supernatant was filtered through a 0.45 μm filter. The recovered VRPs were stored at either 4°C or -80°C.