Construction of eukaryotic expression vector of mouse microRNA miR-21 and identification its expression activity in 293 cells
10.11659/jjssx.1672-5042.201402001
- VernacularTitle:小鼠微小RNA miR-21真核表达载体构建及其在293细胞的活性表达
- Author:
Shuang LONG
;
Yongping SU
;
Jiong REN
;
Huiqin SUN
;
Tao WANG
- Publication Type:Journal Article
- Keywords:
miRNAs;
miR-21;
eukaryotic expression vector;
293 cells
- From:
Journal of Regional Anatomy and Operative Surgery
2014;(2):111-114
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression vector for mouse microRNA miR-21 and identification its expression activ-ity in 293 cells. Methods The genomic sequence containing pre-miR-21 was amplified from mouse genomic DNA by PCR and cloned into the pRC/CMV plasmid. The constructed recombinant plasmid pRC/CMV-mmu-miR-21 was transfected to 293 cells by lipofectamine 2000, and the stably transfected cells were screened with G418,from which total RNA was extracted for detecting the expression of mature miR-21 by northern blot. In the meantime,a luciferase report plasmid examing the activity of miR-21 named pmiR-21-Luc reporter was also construc-ted,and luciferase activity analysis indicated the product of pRC/CMV-mmu-miR-21 indeed had biological activity. Results Both restriction enzyme digestion analysis and sequencing proved the recombinant plasmids were constructed correctly. The miR-21 was highly expressed in the screened clones of 293 cells and it had good biological activity. Conclusion The eukaryotic expression plasmid of mouse miR-21 was successfully constructed,which laid the foundation of further investigation of the role of miR-21 during skin wound healing.
