Lentivirus titer kit

For production of lentiviral particles carrying lem8 or its mutants, the gfp-lem8 fusion was inserted into pCDH-CMV-MCS-EF1a-Puro (System Biosciences, cat# CD510B-1). The plasmids were cotransfected with pMD2.G (gift from Dr. Didier Trono, Addgene #12259) and psPAX2 (gift from Dr. Didier Trono, Addgene #12260) into HEK293T cells grown to about 70% confluence. Supernatant was collected after 48-hr incubation, followed by filtration with 0.45-μm syringe filters. After measuring the titers using qPCR with the Lentivirus Titer Kit (abm, cat# LV900), the packed lentiviral particles were used to infect newly prepared HEK293T cells at an MOI of 10. After incubation for 2 days, cells were sorted by BD Influx cell sorter to establish cell lines stably expressing the gene of interest.

Human 293T cells were used for lentiviral packaging. The lentiviral vector pLenti SV40T purchased from Applied Biological Materials (Abm, Vancouver, BC, Canada) Inc., lentiviral package vector 2nd Generation Packaging Mix (Abm, Vancouver, BC, Canada), and Lentifectin™ Transfection Reagent (Abm, Vancouver, BC, Canada) were co-transfected into human 293T cells to produce lentiviral particles. The medium was collected at 48 h and filtered with a 0.45-μm filter (Millipore, Billerica, MA, USA). The viral supernatants were mixed with 60% 5 × PEG8000 and centrifuged at 10,000× g for 4 h. After discarding the supernatant, sedimentary lentiviral particles were resuspended in DMEM/F12 medium, and the viral titer was determined by a Lentivirus Titer Kit (Abm, Vancouver, BC, Canada). Primary duck intestinal epithelial cells were infected with lentivirus at a multiplicity of infection of 10 and cultured at 37 °C for 48 h; the clonal population was selected by the ability of the immortalized cells confirmed by continuous culture past 20 passages to survive senescence [23 (link)]. Immortalized cells were confirmed by continuous culture past 30 passages and subsequently referred to as IDECs.