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Bratislava Medical Journal 2016 Bratislava Medical Journal Vol.117, No.5, p.272-275, 2016
Bratislava Medical Journal 2016 Bratislava Medical Journal Vol.117, No.5, p.272-275, 2016
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Bratislava Medical Journal Vol.117, No.5, p.272-275, 2016 |
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| Title: Construction of a Hep-2 cell line stably transfected with Livin shRNA | ||
| Author: S. L. Wang, W. T. Deng, G. F. Wen, C. W. Li, Y. J. Zeng | ||
| Abstract: OBJECTIVE: The aim of this study was to construct a eukaryotic expression plasmid with a short hairpin RNA (shRNA) targeting Livin in order to obtain a stably transfected Hep-2 cell line with a reduced expression of Livin. METHODS: The shRNA targeting Livin mRNA was designed, and a shRNA plasmid and a negative control plasmid were constructed. After amplification in E. coli, restriction endonuclease digestion and sequence confirmation, the plasmids were transfected into Hep-2 cells using Lipofectamine 2000. The stably transfected cell line was screened using G418, and inhibition of Livin mRNA and protein levels were detected using real-time PCR and western blotting, respectively. RESULTS: pGenesil-Livin-shRNA eukaryotic expression plasmid was successfully constructed and identified by sequencing. Green fluorescent protein (GFP) expression was observed in Hep-2 cells transfected with shRNA plasmids by fluorescence microscopy. The expression levels of Livin mRNA and protein decreased significantly in Hep-2 cells transfected with the shRNA recombinant plasmid. The mRNA level was reduced by 47.17 %, and the protein level was reduced by 34.25 %. CONCLUSION: The shRNA eukaryotic expression plasmid targeting Livin was successfully constructed, which could significantly inhibit the expression of Livin in laryngeal cancer Hep-2 cells. This provides a basis for future research on the function of Livin in Hep-2 cells, and gene therapy for laryngeal cancer (Fig. 5, Ref. 10). |
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| Keywords: apoptosis regulatory proteins, cell culture, RNA interference, livin gene | ||
| Published online: 13-May-2016 | ||
| Year: 2016, Volume: 117, Issue: 5 | Page From: 272, Page To: 275 | |
| doi:10.4149/BLL_2016_053 |
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