The sense strand of IRF4 shRNA and EBNA3C shRNA sequences are 5′- tcgagtgctgttgacagtgagcgaGCATGAACCTGGAGGGCGGtagtgaagccacagatgtaCCGCCCTCCAGGTTCATGC gtgcctactgcctcggaa-3′ [43] (link) and 5′- tcgagtgctgttgacagtgagcgaCCATATACCGCAAGGAATAtagtgaagccacagatgtaTATTCCTTGCGGTATATGGgtgcctactgcctcggaa-3′ [64] respectively. Here, upper-case letters designate either IRF4 or EBNA3C target sequences and lower-case letters specify hairpin and sequences which are required for the directional cloning in pGIPZ vector. These single stranded oligonucleotides were individually cloned into the pGIPZ vector using XhoI and MluI restriction sites. Also, a control shRNA sequence; 5′-TCTCGCTTGGGCGAGAGTAAG-3′ (Dharmacon Research, Chicago, IL) was used to make Sh-Ctrl vector which lack the complementary sequences in the human genome.
For production of lentivirus, 2×106 HEK 293T cells were grown in DMEM media with 10% FBS for 24 hrs prior to transfection. Total 20 µg of plasmid expression vector was used for the transfection of each set, including 1.5 µg of pCMV-VSV-G, 3 µg of pRSV-REV, 5 µg of pMDLg/Prre (Addgene, Inc., Cambridge, MA), and 10.5 µg of lentiviral vector plasmid. For precipitation, plasmids were added to a final volume of 438 µl of sterile H20 and 62 µl of 2 M CaCl2, and solutions mixed well, then 500 µl of 2×HEPES-buffered saline added. Each transfection set was incubated at room temperature for 30 min. Before transfection, chloroquine was added to the 10 ml of media with a final concentration of 25 µM for 5 min. The media was replaced with DMEM supplemented with 10% FBS and 10 mM HEPES, and 10 mM sodium butyrate after 12 hrs of incubation. Again, the media was replaced after 10 hrs by DMEM supplemented with 10% FBS with 10 mM HEPES. To collect virus, the conditioned media was collected four times at 12 hrs interval. Conditioned medium was filtered through cellulose acetate filters (0.45 µm) and stored in ice. The virus was concentrated by centrifuging the medium at 70,000×g for 2.5 hrs. The concentrated virus was re-suspended in RPMI medium and the virus used to infect 106 LCL1 cells with Polybrene as 20 µM/ml concentration. After 72 hrs of incubation, puromycin antibiotic was added as 2 µg/ml concentration for selection. To check the rate of selection, GFP-immunofluorescence was observed by Olympus 1X71 microscope with 560 nm excitation and 645 nm emission filters. Puromycin selected cells were grown up to 80% confluence and the expression levels of target proteins were checked by western blot analysis.
For production of lentivirus, 2×106 HEK 293T cells were grown in DMEM media with 10% FBS for 24 hrs prior to transfection. Total 20 µg of plasmid expression vector was used for the transfection of each set, including 1.5 µg of pCMV-VSV-G, 3 µg of pRSV-REV, 5 µg of pMDLg/Prre (Addgene, Inc., Cambridge, MA), and 10.5 µg of lentiviral vector plasmid. For precipitation, plasmids were added to a final volume of 438 µl of sterile H20 and 62 µl of 2 M CaCl2, and solutions mixed well, then 500 µl of 2×HEPES-buffered saline added. Each transfection set was incubated at room temperature for 30 min. Before transfection, chloroquine was added to the 10 ml of media with a final concentration of 25 µM for 5 min. The media was replaced with DMEM supplemented with 10% FBS and 10 mM HEPES, and 10 mM sodium butyrate after 12 hrs of incubation. Again, the media was replaced after 10 hrs by DMEM supplemented with 10% FBS with 10 mM HEPES. To collect virus, the conditioned media was collected four times at 12 hrs interval. Conditioned medium was filtered through cellulose acetate filters (0.45 µm) and stored in ice. The virus was concentrated by centrifuging the medium at 70,000×g for 2.5 hrs. The concentrated virus was re-suspended in RPMI medium and the virus used to infect 106 LCL1 cells with Polybrene as 20 µM/ml concentration. After 72 hrs of incubation, puromycin antibiotic was added as 2 µg/ml concentration for selection. To check the rate of selection, GFP-immunofluorescence was observed by Olympus 1X71 microscope with 560 nm excitation and 645 nm emission filters. Puromycin selected cells were grown up to 80% confluence and the expression levels of target proteins were checked by western blot analysis.
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