Cloning of CGI-100 gene and reconstruction of its eukaryotic expressive vector / 重庆医科大学学报

Objective:To clone human CGI-100 gene and reconstruct its eukaryotic expressive vector for further investigation.Methods:The full coding domain sequence of human CGI-100 gene was cloned from human leukemic K562 cells with RT-PCR and was sub-cloned into pMD18-T Simple vector.After confirmed by DNA sequencing,the targeted DNA fragment,digested with BamHⅠand PstⅠ,was directionally cloned into eukaryotic expressive plasmid pIRES2-EGFP,then,the reconstructed plasmid was identified with enzyme digestion,PCR and sequencing.Results:It was demonstrated that the full coding domain sequence of human CGI-100 gene was accurately cloned into digestion sites between BamHⅠand PstⅠin pIRES2-EGFP without mutation and transposition.Conclusion:The reconstruction and verifying of eukaryotic expressive plasmid containing CGI-100 gene are successful,which establishes the foundation of further investigation.

Construction and identification of the RNA interference system targeting CGI-100 gene / 重庆医科大学学报

Objective:To construct the expression vector of RNA interference(RNAi)system for CGI-100 gene and identify its inhibition efficiency.Methods:Three oligonucleotides targeting CGI-100 gene were devised and chemically synthesized,annealed to form double-strand DNA and inserted into plasmid PGenesil-1 whose segments were digested by BamHⅠ and HindⅢ.The recombinant vector were then transfected into K562 cells with lipofectamineTM 2000.CGI-100 expresstion in the transfected cells was assayed by RT-PCR and dot blot.Results:The recombinant plasmids of PGenesil-1-CGI-100 were successfully constructed by DNA sequencing analysis.The first of PGenesil-1-CGI-100 had the highest inhibition efficiency in mRNA levels,up to 54%.Conclusion:The RNA interference system targeting CGI-100 gene were successfully constructed and a interference vector with a higher inhibition efficiency was screened,which provides the foundation for further investigation.