Construction and identification of the small interfering RNA eukaryotic expression vectors of human ClC-2 gene / 医学研究生学报

ABSTRACT

Objective:To construct the small interfering RNA(siRNA) eukaryotic expression vectors of human ClC-2 gene. Methods: According to the program and principles of DEQOR about designing siRNA,two pairs ClC-2 mRNA-targeted hairpin siRNA were devised,and the two pairs complemantary oligonucleotide strands of DNA fragments that encoded the above siRNA were synthesized by chemosynthesis.After annealing of the complementary strands,the DNA fragments were connected to the polyclone sites of plasmid eukaryotic expression vector pSUPER.puro that was cut by restriction endonuclease BglⅡ and HindⅢ,followed by transformation,amplification and plasmid extraction in E.coli,and finally,the two recombinant plasmids were identified by agarose gel electrophoresis by means of cutting with EcoRⅠand HindⅢ and by DNA sequence analysis.The plasmids were transfected transiently into human glioma cell line BT-325 cells by Lipofectamine~(TM)2000.The mRNA expression of ClC-2 gene was detected by(RT-PCR.) Results: The connections between the DNA fragments encoding ClC-2-targeted siRNA and the pSUPER.puro plasmid were correct,as confirmed by agarose gel electrophoresis and DNA sequencing(analysis.) ClC-2 mRNA expression of the BT-325 cells transfected two recombinant vectors was(significantly) decreased.Conclusion: The two RNAi recombinant vectors of human ClC-2 gene were successfully constructed.They were named pSUPER.puro-siClC-21 and pSUPER.puro-siClC-22,respectively.This laid the groundwork for future research about ClC-2 gene affecting invasion and migration of human glioma cells.