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[Construction and application of inducible artificial microRNA expression vector targeting eIF3g gene]
Li-Sha Chu1, Na Han, Xiang-Yang Song
1Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou 310016, China. 2008chulisha@163.com
Aim:
To construct an inducible artificial microRNA expression vector targeting eIF3g gene and use it to inhibit the expression of eIF3g in K562 cells.
Methods:
The microRNA targeting human eIF3g was designed and obtained by PCR. After confirmed by sequencing, the microRNA was cloned into the pRevTRE2 plasmid. The Tet-off plasmids were transfected into K562 cells and selected for the stable tetracycline inducible K562/Tet-off transfectants. The pRevTRE2-eIF3g-miRNA plasmid was then transfected into stable K562/Tet-off transfectants and the expression of eIF3g was detected by Western blot.
Results:
The microRNA targeting eIF3g was confirmed by sequencing. GFP fluorescence assay showed that the stable transfectants of Tet-off plasmids were under tetracycline control. Western blot results showed that the stable transfectants of pRevTRE2-eIF3g-microRNA inhibited eIF3g expression under the regulation of tetracycline.
Conclusion:
The tetracycline-inducible artificial microRNA expression vector targeting eIF3g gene has been successfully constructed and effectively inhibits eIF3g expression in K562 cells.
