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siRNA suppresses the green fluorescent protein (GFP) gene expression in human umbilical vein endothelial cells / 中国病理生理杂志
Chinese Journal of Pathophysiology ; (12)1986.
Artículo en Chino | WPRIM | ID: wpr-526878
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.

Texto completo: Disponible Índice: WPRIM (Pacífico Occidental) Idioma: Chino Revista: Chinese Journal of Pathophysiology Año: 1986 Tipo del documento: Artículo

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Texto completo: Disponible Índice: WPRIM (Pacífico Occidental) Idioma: Chino Revista: Chinese Journal of Pathophysiology Año: 1986 Tipo del documento: Artículo