Establishment of a canine WRD cell line with stable p53 knockdown by lentivirus-mediated p53 silencing
10.3969/j.issn.1673-4254.2014.12.03
- VernacularTitle:慢病毒介导的犬WRD细胞p53基因沉默及稳定感染细胞系的建立
- Author:
Fang LI
1
;
Yan YAN
;
Qian ZHANG
;
Jianning LI
;
Qing YAO
;
Yujing GAO
;
Yi YANG
;
Yuning SUN
Author Information
1. 宁夏医科大学总院呼吸与危重医学科
- Keywords:
p53;
RNA interference;
lentivirus vector;
WRD cells
- From:
Journal of Southern Medical University
2014;(12):1721-1727
- CountryChina
- Language:Chinese
-
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.
