Jetpei macrophage dna transfection reagent

NF-κB activity assay was performed using NF-κB luciferase reporter kit (BPS Biosci-ence, Cat#: 60614) according to the manufacturer’s instruction. Macrophages with or without various treatments were co-transfected with NF-κB luciferase reporter and pRL-TK renilla luciferase reporter by jetPEI-Macrophage DNA Transfection Reagent (Polyplus-transfection, Cat#: 101000043). The luciferase activity was measured using a Dual Luciferase Assay System (Berthold Technologies).

The I226R gene was deleted from the ASFV SY18 genome and replaced with an eGFP gene by homologous recombination between the SY18 virus and a recombinant plasmid comprising the eGFP expression cassette under the control of the p72 promoter and two sequences flanking the I226R gene of about 1,200 bp. The recombination plasmid of about 2 μg was transfected into PAMs in a 12-well plate using jetPEI-Macrophage DNA transfection reagent (Polyplus). After 4 h, the transfected cells were infected with ASFV SY18 at an MOI of 3 and continued to be cultured at 37°C under 5% CO₂. The cells exhibiting fluorescence were picked out under a fluorescence microscope, further screened, and purified by limiting dilution. The final recombinant virus was amplified in PAMs. The purity of SY18ΔI226R was determined by PCR. The forward and reverse primers 5′-CTACGCCGAACTATTCAGA-3′ and 5′-GTCACCAACAACAGGATAAC-3′ were designed based on the sequences in the middle of the I226R coding region and the right flanking region, respectively, using Primer Premier 6 software. A 492-bp fragment could be amplified if parental SY18 exists.

BMDMs were cultured in 24-well plates with differentiated medium and transduced with ctrl-, M14-WT, M14-Mut, M3-WT, M3-Mut or Kat2b-lentivirus for 48 h. After transduction, BMDMs were co-transfected with 500 ng pGL3-Promoter (control), pGL3-Spi2a-WT or pGL3-Spi2a-Mut and pRL-TK renilla luciferase reporter using jetPEI-Macrophage DNA Transfection Reagent (Polyplus-transfection, Cat#: 101000043). 24 h after transfection, BMDMs were lysed and luciferase activities were measured by a Bio-Glo Luciferase Assay System kit (Promega, Cat#: G7941) according to the manufacturer’s instruction. In another experiment, BMDMs were treated with LPS for 8 h after 24-h luciferase reporter vector transfection, and then luciferase activities were detected as described above.