Construction and identification of over-expression vector and RNA interference vectors of receptor for activated C-kinase 1
ZHANG Li
JIA Xiong-fei
NIU Hua
ZHENG Rui
ZHANG Wang-long
MAO Xiao-qin
Abstract：Objective To construct and identify the over-expression and interference eukaryotic expression vectors of receptor for activated C-kinase 1 (RACK1).Methods The RACK1 gene full-length reading frame was amplified from the human hepatoma cell line Huh-7.5.1 by RT-PCR.Then we amplified the corresponding CDS by nested PCR and cloned it into PIRES2-EGFP.The monoclone was identified by PCR and DNA sequencing .At the same time, we designed and syn-thesized complementary DNA sequences of 2 pairs of short hairpin structure and a pair of negative control sequence with hu-man RACK1 gene.After annealing, they were linked into restriction enzyme digested RNAi-Ready pSIREN-RetroQ-Zs-Green vector , and then identified by enzyme digestion analysis and DNA sequencing .The constructed over-expression vec-tor and interference vectors were transfected into HUVEC cell line by liposome .Results The size of two pairs of primers amplified by PCR sequences was completely true .A total of 954 nt oligonucleotides were successfully inserted into the vec-tor PIRES2-EGFP.The inserted sequences of RNA interference vectors pRetroQ /RACK1-1, pRetroQ/RACK1-2 and pRet-roQ/HK were consistent with our expectations .The EGFP and GFP could be seen in the transfected HUVEC cell line by fluorescence microscopy .Conclusion The over-expression vector and siRNA expression vectors of RACK 1 are successful-ly constructed , and can be introduced into HUVEC cell line .
Keywords：receptor for activated C-kinase 1over-expression vectorinterference vectorRNA interference
Publication Date：2014-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 11-14 )