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Published on: August 25, 2021
[Construction and expression of eukaryotic expression vector of a novel apoptosis-related mouse gene, mPNAS-4 and its
Zhu Yuan1, Fei Yan, Xin-Yu Zhao
1School of Life Science, Sichuan University, Chengdu 610064, China. yuanzhujyx@gmail.com
Aim:
To construct eukaryotic vector of a novel apoptosis-related gene from mouse (mPNAS-4) and overexpress it in mouse Lewis lung carcinoma(LL2) cell line. To explore the apoptosis of the tumor cells induced by overexpressed mPNAS-4 in LL2 cells via transfection.
Methods:
RT-PCR was applied to amplify the encoding region of mPNAS-4 gene from the mouse liver. The cDNA was cloned into the eukaryotic expression vector pcDNA3.1(+) and the resulting recombinant expression plasmid pcDNA3.1(+)-mPNAS-4 was then transfected into mouse Lewis lung carcinoma (LL2) cells through Lipofectamine 2000. Overexpression of mPNAS-4 in the transfected cells and proliferation of the transfected cells was detected by RT-PCR and MTT assay,respectively, while the apoptosis was analysed by FCM and DNA ladder.
Results:
The recombinant eukaryotic expression vector pcDNA3.1(+)-mPNAS-4 was successfully constructed.The expression of mPNAS-4 at the mRNA level in LL2 cells transfected with pcDNA3.1(+)-mPNAS-4 was up-regulated significantly. The proliferation of LL2 cells was inhibited.
Conclusion:
Overexpression of mPNAS-4 may have apoptotic effects and therefore inhibit the proliferation of LL2 cells.
Insights
Overexpressing the mouse PNAS-4 (mPNAS-4) gene in lung cancer cells induced apoptosis and inhibited tumor cell proliferation. This study successfully constructed a vector for mPNAS-4 gene delivery and confirmed its effects in vitro.
Area of Science:
- Molecular Biology
- Cancer Research
Background:
- Apoptosis plays a crucial role in regulating cell death and tumor suppression.
- Identifying novel genes involved in apoptosis is essential for cancer therapy development.
Purpose of the Study:
- To construct a eukaryotic expression vector for the mouse apoptosis-related gene mPNAS-4.
- To investigate the effects of mPNAS-4 overexpression on apoptosis and proliferation in mouse Lewis lung carcinoma (LL2) cells.
Main Methods:
- Amplification and cloning of the mPNAS-4 gene into the pcDNA3.1(+) vector.
- Transfection of the recombinant plasmid into LL2 cells using Lipofectamine 2000.
- Analysis of mPNAS-4 expression, cell proliferation, and apoptosis using RT-PCR, MTT assay, FCM, and DNA laddering.
Main Results:
- Successful construction of the pcDNA3.1(+)-mPNAS-4 recombinant vector.
- Significant upregulation of mPNAS-4 mRNA expression in transfected LL2 cells.
- Inhibition of LL2 cell proliferation observed.
Conclusions:
- Overexpression of mPNAS-4 induces apoptosis in LL2 cells.
- mPNAS-4 exhibits potential as a therapeutic agent for inhibiting lung cancer cell proliferation.
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