OBJECTIVETo establish a canine cell line with p53 gene knockdown by lentivirus- mediated RNA interference (RNAi).

METHODSFour pairs of oligonucleotide sequences of p53 gene were synthesized and cloned into the pGMLV-SC5 RNAi vector. Following confirmation by PCR and DNA sequencing, the recombinant pGMLV-p53 plasmids and packaging mix vectors were packaged into mature lentivirus to infect 293T cells. The supernatant of the infected cells was harvested to infect WRD cells, in which p53 expression was detected using Western blotting and real-time PCR. The lentivirus with the strongest p53⁻ silencing effect was packaged for infecting WRD cells at the optimal multiplicity of infection (MOI) to establish a stably infected cell line (WRD/p53⁻) screened using puromycin.

RESULTSThe lentivirus carrying p53 shRNA was constructed successfully and a virus titer of 1 × 10⁹ TU/ml. The 4 pGMLV-p53 plasmids all produced strong p53 interference affects, and pGMLV- p53A1 with the strongest effect. The stably infected cells line WRD/p53⁻ was established successfully using pGMLV-p53A1 plasmid.

CONCLUSIONThe canine cell line WRD/p53⁻ with stable lentivirus-mediated p53 silencing has been established successfully.