Construction of vectors generating Nurr1 shRNA and their silencing effect in cultured dopaminergic cell lines / 中华神经科杂志

ABSTRACT

Objective To construct the nuclear receptor-related factor 1 ( Nurr1) specific short-hairpin RNA (shRNA) expressing vectors and study their effects on the expression of endogenous genes in MN9D dopaminergic cell lines. Methods Two RNAi recombinant plasmids (named pSC-N1 and pSC-N2) targeting at Nurrl gene were constructed. The purified plasmids were identified by DNA sequencing. After being transfected into MN9D dopaminergic cell lines with Lipofectamine?2000, the silencing effects of Nurrl shRNA expressing vectors on the expression of Nurrl mRNA and protein in MN9D cells were detected using real time fluorescence quantitative PCR and Western blot Results DNA sequencing confirmed that the Nurrl shRNA expressing vectors were constructed successfully. Nurrl mRNA expression in MN9D cells was specifically suppressed after the transfection of pSC-Nl and pSC-N2 by 62.3% and 45.6% respectively. Moreover, the expression of Nurrl protein was significantly suppressed by 57.4% and 72. 0% respectively. The control vectors had no silencing effect Conclusions The vectors pSC-Nl and pSC-N2 have been successfully constructed and can specifically suppress the expression of Nurrl mRNA and protein. Nurrl specific shRNA expressing vector may provide a novel applicable strategy for the study of the function of the genes associated with Parkinson disease and the development of dopaminergic neuron.