Abstract: Objective To establish a canine cell line with p53 gene knockdown by lentivirus- mediated RNA interference (RNAi).
Methods Four 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. Results The 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.
Conclusion The canine cell line WRD/p53- with stable lentivirus-mediated p53 silencing has been established successfully.