[Construction and expression of eukaryotic expression vector of a novel apoptosis-related mouse gene, mPNAS-4 and its antitumor activity in vitro].
OBJECTIVE
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.


CONCLUSIONS
Overexpression of mPNAS-4 may have apoptotic effects and therefore inhibit the proliferation of LL2 cells.
Yuan
Zhu
Z
School of Life Science, Sichuan University, Chengdu 610064, China. yuanzhujyx@gmail.com
Yan
Fei
F
Zhao
Xin-Yu
XY
Deng
Hong-Xin
HX
Li
Jiong
J
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D053938:DNA Fragmentation; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins