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Published on: December 12, 2017
[Construction of a GFP/Puro double-labeled lentiviral vector containing CK8 interfering RNA and its effect on cell
Yanchao Jin1, Ronghua Yin, Weiwei Zheng
1Department of Pharmaceutical Engineering, Tianjin University, Tianjin 300072, China.
Objective:
To construct a GFP/Puro double-labeled lentiviral expression vector for CK8 silencing and assess the effects of CK8 silencing on cell apoptosis.
Methods:
The siRNA sequences of CK8 were inserted into the lentiviral expression vector GV248 and transfected into 293T cells with the packaging plasmids PMD and SPA. The lentivirus was collected at 24 and 36 h post-transfection. Flow cytometry was used to detect the virus titer and the positive cells were selected with puromycin. The knockdown of CK8 was examined by Western blotting. The effect of CK8 down-regulation on cell apoptosis induced by cisplatin was detected with Annexin V/PI staining.
Results And Conclusion:
We successfully constructed CK8 interference lentiviral vector and obtained a stable cell line with CK8 knock-down that was sensitive to cisplatin-induced apoptosis.
Insights
Researchers developed a lentiviral vector to silence Keratin 8 (CK8) and found that CK8 knockdown increases sensitivity to cisplatin-induced apoptosis in cancer cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Keratin 8 (CK8) is implicated in various cellular processes.
- Understanding CK8's role in apoptosis is crucial for cancer therapy.
- Developing tools for gene silencing is essential for functional studies.
Purpose of the Study:
- To construct a dual-labeled lentiviral expression vector for CK8 gene silencing.
- To evaluate the impact of CK8 knockdown on cisplatin-induced apoptosis.
Main Methods:
- Insertion of CK8 siRNA into the GV248 lentiviral vector.
- Transfection of 293T cells and lentivirus production.
- Flow cytometry for virus titration and puromycin selection.
- Western blotting to confirm CK8 knockdown.
- Annexin V/PI staining to assess apoptosis.
Main Results:
- Successful construction of a GFP/Puro double-labeled lentiviral vector for CK8.
- Generation of a stable cell line with significantly reduced CK8 expression.
- Demonstrated increased sensitivity to cisplatin-induced apoptosis in CK8-silenced cells.
Conclusions:
- A functional CK8 interference lentiviral vector was successfully created.
- A stable CK8 knockdown cell line was established.
- CK8 downregulation enhances cisplatin-induced apoptosis, suggesting a therapeutic target.
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