siRNA suppresses the green fluorescent protein (GFP) gene expression in human umbilical vein endothelial cells
- VernacularTitle:用小双链RNA抑制转化入HUVEC中绿色荧光蛋白基因的表达
- Author:
Zhixin SHAN
;
Qiuxiong LIN
;
Xiyong YU
;
Chunyu DENG
;
Meng ZHENG
;
Honghong TAN
;
Yongheng FU
;
Min YANG
;
Shuguang LIN
- Publication Type:Journal Article
- Keywords:
RNA interference;
Green fluorescent protein;
Human umbilical vein endothelial cells;
Liposomes
- From:
Chinese Journal of Pathophysiology
1986;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To establish human umbilical vein endothelial cells (HUVECs) to express green fluorescent protein (GFP), and to study the suppression of GFP by siRNA in HUVECs. METHODS: Using lipofectamine 2000 to transform plasmid pN_3-EGFP encoding GFP into HUVECs. The HUVEC containing pN_3-EGFP, named HUVEC-GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control-siRNA used as control were synthesized. The siRNAs were transfected into HUVEC-GFP with oligofectamine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC-GFP were determined. RESULTS: The HUVEC-GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC-GFP transfected with GFPsiRNA. The results of RT-PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC-GFP transfected with control siRNA. CONCLUSION: The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.
