Construction and identification of small interfering RNA expression vector targeting EOLA1 gene
- VernacularTitle:EOLA1基因小干扰RNA载体构建及其干扰效应检测
- Author:
Yueming LIU
;
Zongcheng YANG
;
Ziwen LIANG
- Publication Type:Journal Article
- Keywords:
RNA interference;
gene, EOLA1;
transfection
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct small interfering RNA(siRNA) expression vectors targeting EOLA1 (Endothelial-overexpressed lipopolysaccharide-associated factor 1) gene, and to assay their effects on the expression of EOLA1. Methods GFP-EOLA1 fusion protein expressive vector pEGFP-N2/EOLA1 was constructed and transfected into ECV304 cells. The transfected cells were cultured in M199 containing G418 (400?g/ml) for 5 weeks to screen the cell line stably expressing GFP-EOLA1 fusion protein. For interfering EOLA1 target gene, complementary oligonucleotides were desiqned and synthesized at different sites. The oligonucleotides were inserted into pSinencer3.1/H1 plasmid. Then, the vector was transfected into the ECV304 cells stably expressing GFP-EOLA1 fusion protein and the inhibiting affection to target gene EOLA1 was identitied by the observation of the green fluorescence in transfected cells with inverted fluorescent microscope and Western immunoblot assay. Results The ECV304 cell line stably expressing GFP-EOLA1 fusion protein was constructed, and the siRNA vector of EOLA1 can knockdown EOLA1 gene expression specifically. Conclusion The siRNA vectors specifically interfering EOLA1 expression were constructed successfully, which would be useful in the study of function of EOLA1 gene.
