Construction and identification of a eukaryotic expression vector for the small interfering RNA targeting nucleostemin gene / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for the small interfering RNA (siRNA) targeting nucleostemin (NS) gene.</p><p><b>METHODS</b>The siRNA targeting NS gene was designed according to the sequence of NS mRNA available in GenBank. Three siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into plasmid pRNAT-U6.1 for constructing the recombinant plasmids, which were transformed into E.coli DH5alpha strain. The plasmids, after identification by PCR and DNA sequencing, were transfected into EC9706 cell line via liposome, and the mRNA and protein expressions of NS gene in the cells were determined by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Three recombinant plasmids were identified by PCR and sequence analysis, the results of which showed correct insertion of the designed sequences in the plasmids. RT-PCR and Western blotting showed substantially decreased mRNA and protein expressions of NS gene in the transfected cells.</p><p><b>CONCLUSION</b>The recombinant plasmid expressing the siRNA targeting NS gene has been successfully constructed, which provides the basis for studying RNA interference of the NS gene.</p>