Abstract
Objective
To construct eukaryotic expression plasmid pEGFP-N1-WWOX and transiently express it in SMMC-7721 cells.
Methods
Total mRNA was extracted from normal human liver tissue. RT-PCR was used to amplify the aimed segments WWOX cDNA which was then digested with HindIII and BamHI and inserted into a eukaryotic expression plasmid pEGFP-N1 to construct pEGFP-N1-WWOX. The constructed plasmid was transfected into SMMC-7721 cells by lipofectamine 2000 — mediated transfer method. The expression of WWOX in transfected SMMC-7721 cells was detected 24, 36 and 48 h post-transfection with fluorescence microscope and the expression level of WWOX mRNA in transfected SMMC-7721 cells was assay by using RT-PCR. The change of WWOX expression and cell proliferation rates were detected by immunocytochemistry and MTT methods respectively.
Results
The results showed pEGFP-N1-WWOX was successfully constructed and expressed transiently in SMMC-7721 cells. At 48th hour post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected, while no green fluorescence was detected in the control group. In SMMC-7721 cells transfected with pEGFP-N1-WWOX a high level of porcine WWOX was detected. WWOX expressed by transfected cells could significantly inhibit the proliferation of SMMC-7721 cells.
Conclusion
pEGFP-N1-WWOX was expressed successfully in SMMC-7721 cells, which suggested that might be used as a new therapeutic method for liver cancer.
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Liu, F., Li, X. & Yi, J. Construction of eukaryotic expression plasmid pEGFP-N1-WWOX and its transient expression in SMMC-7721 cells. Chin. -Ger. J. Clin. Oncol. 8, 61–64 (2009). https://doi.org/10.1007/s10330-008-0155-3
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DOI: https://doi.org/10.1007/s10330-008-0155-3