Construction and identification of small interfering RNA expression plasmid target to angiotensinogen / 中国组织工程研究

ABSTRACT

BACKGROUND:In mammalian cells,introduction of double-stranded small interfering RNA(19-25 bp)can cleave and destroy the cognate RNA,which can result in suppression of gene expression.OBJECTIVE:To construct siRNA expression plasmid for interference angiotensinogen(AGT),thereby,to resist AGT expression in adipose cells.METHODs:The mRNA sequence of AGT gene was searched from NCBI(NM000029).Utilize of GenScript siRNA technology,AGT-siRNA oliaonucletides were chemically synthesized and inserted into pRNAT-U6 1/Neo vector after annealing,then transformed into TOP10.The recombinant plasmid was identified by restriction endonuclease and DNA sequencing.RESULTS AND CONCLUS1ONThe recombinant plasmid psiRNAT-U6.1/Neo-AGT was obtained by connecting 19 bp segment containing AGT-mRNA sequence to pRNAT-U6.1/Neo After EcoR Ⅰ and Hind Ⅲ digestion.351 bp segment was obtained from empty vector.and 397 bp fragment band was obtained form recombinant plasmid,which was coincidence to the expectation.DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pRNAT-U6.1/Neo without base mutation.The interference vector psiRNAT-U6.1/Neo-AGT was successfully constructed.