Basic & Clinical Medicine ›› 2015, Vol. 35 ›› Issue (2): 218-223.

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Establishment and identification of HUVEC cell strains with over-expression and low expression of RACK1

  

  • Received:2014-03-31 Revised:2014-09-27 Online:2015-02-05 Published:2015-01-23

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

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