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Published on: March 5, 2013
[Establishment of a canine WRD cell line with stable p53 knockdown by lentivirus-mediated p53 silencing]
1Deptartment of Respiratory Medicine, General Hospital of Ningxia Medical University, Yinchuan 750004, China.E-mail: dreamernew@gmail.com.
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.
Insights
Researchers successfully created a canine cell line with a silenced p53 gene using lentivirus-mediated RNA interference (RNAi). This new WRD/p53⁻ cell line offers a valuable tool for cancer research and drug development.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- The p53 gene is a critical tumor suppressor involved in cell cycle regulation and apoptosis.
- Developing genetically modified cell lines is essential for studying gene function and disease mechanisms.
Purpose of the Study:
- To establish a canine cell line with stable knockdown of the p53 gene using lentivirus-mediated RNA interference (RNAi).
Main Methods:
- Synthesized and cloned p53 gene-targeting oligonucleotide sequences into an RNAi vector.
- Packaged recombinant plasmids into lentivirus to infect 293T and WRD cells.
- Confirmed p53 knockdown using Western blotting and real-time PCR, establishing the WRD/p53⁻ cell line via puromycin screening.
Main Results:
- Successfully constructed lentivirus carrying p53 shRNA with a titer of 1 × 10⁹ TU/ml.
- Identified pGMLV-p53A1 as the most effective plasmid for p53 interference.
- Established the stable canine WRD/p53⁻ cell line with effective p53 gene silencing.
Conclusions:
- Successfully established a canine cell line (WRD/p53⁻) with stable p53 gene silencing via lentivirus-mediated RNAi.
- This cell line serves as a valuable model for investigating p53 functions in canine systems.
