关键词: RACK1, 过表达, RNA干扰, HUVEC细胞株

Abstract: Objective To establish several human umbilical vein endothelial cell(HUVEC) strains with over-expression or low expression of Receptor for Activated C Kinase 1 (RACK1), which will provide effective means for future studying the function of RACK1 in arrhythmia. Methods The full-length cDNA sequence of RACK1 gene was amplified and inserted into pIRES2-EGFP. At the same time, designed and synthesised complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence, then subcloned into the plasmid pGenesil-1. The HUVEC cells were transfected with these plasmids and screened by using G418. And the expression of RACK1 mRNA and protein in the cells were assayed by qRT-PCR and Western blot, respectively. Results RACK1 eukaryotic expression vector and siRNA expression vectors of RACK1 were constructed successfully. After a 48 h transfection of HUVEC cells with the recombinant vectors and G418 selection, the positive cell clones were obtained. qRT-PCR and Western blot showed that over-expression vector and interference vectors could effectively enhance and knock-down RACK1 expression in HUVEC strains. Conclusion HUVEC cell strains with over-expression and low expression of RACK1 have been established successfully.

Key words: RACK1, over-expression, RNA interference, HUVEC strain