Pgfp c shlenti shrna vector

The target sequence of NLRP3 shRNA was
(i) 5^/-AGAGAAGGCAGACCATGTGGATCTAGCCA-3′^/,
(ii) 5^/-CAGTCTGATTCAGGAGAACGAGGTCCTCT-3′^/,
(iii) 5^/-GAGACATTCTCCTGAGCAGCCTCATCAGA-3′^/,
(iv) 5^/- GTACGTGAGAAGCAGATTCCAGTGCATTG-3′^/.
Annealed shRNA synthesized cDNA fragments corresponding to the cDNAs of NLRP3 genes were digested with BamHI and EcoRI and ligated into pLVX vector (HANBIO, Shanghai, China), named as lentivirus expressing NLRP3 shRNA (shNLRP3). Negative control (sh-NC), an ineffective shRNA cassette in the pGFP-C-shLenti shRNA vector plasmid, was purchased from ORIGENE (Rockville, USA). SVA 3D was digested with BamHI and EcoRI and ligated in the LV011-pHBLV-CMV-MCS-3flag-EF1-T2A-Zsgreen-Puro (HANBIO, Shanghai, China). The Lentivector encoding shNLRP3 and Lenti-3D were transfected into HEK293T cells together with psPAX2 and pMD2.G with Lipofectamine 2000. The primers are shown in Table S1. Culture supernatants were harvested at 48 h and 72 h. Supernatants were collected at 48 h and 72 h, filtered with a 0.45 μm filter, and after that centrifuged at 72,000 × g for 120 min at 4°C. BMDM cells, PK-15 cells and pigs were infected with the supernatants containing lentiviral particles. shRNA knockdown efficiencies were assessed by Western blot analysis. The IL-1β level and IL-1β mRNA levels from infected cells and infected pig organs were measured by ELISA and qRT-PCR.

The target sequence of porcine NLRP3 shRNA was 5′-GGTGACCTCATATGACTAA-3′. Annealed short hairpin RNA (shRNA)-synthesized cDNA fragments corresponding to the cDNAs of swine NLRP3 genes were digested with BamHI and EcoRI and ligated into the pLVX vector (HANBIO, Shanghai, China), which was named lentivirus expressing NLRP3 shRNA (shNLRP3). A non-effective shRNA cassette in the pGFP-C-shLenti shRNA vector plasmid was used as the negative control and purchased from ORIGENE (Rockville, MD, USA). The lenti vector encoding shNLRP3 was transfected into HEK293 cells together with psPAX2 and pMD2.G with Lipofectamine 2000 (invitrogen, Waltham, MA, USA). Culture supernatants were harvested at 48 and 72 h, then filtered with a 0.45 μm filter, and centrifuged at 72,000× g for 120 min at 4 °C. PK-15 cells were infected with supernatants containing lentiviral particles. The shRNA knockdown efficiency was assessed by Western blot analysis. The IL-1β level and IL-1β relative mRNA levels in infected PK-15 cells were measured by ELISA (ELISA kit from RayBiotech, Inc., Catalog #: ELP-IL1b) and qRT-PCR (List of primers are given in Table 1).