Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2018-06-15 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To construct a lentiviral vector for delivering short hairpin RNA (shRNA) targeting PAX6 and investigate its effect on the proliferation of glioma U251 cells in vitro. Methods Two small interfering RNA sequences targeting PAX6 gene were designed based on the reported sequence of PAX6 and annealed to form a double-stranded chain, which was inserted into a lentiviral vector to construct the recombinant lentiviral vector shRNA-PAX6. The recombinant vector was infected into U251 cells, and the expression of PAX6 mRNA and protein in the cells was detected by real-time PCR and Western blotting, respectively. The changes in the proliferation of U251 cells after the infection was assessed using MTT assay. Results Double enzyme digestion of the lentiviral vector pLKD-CMV-G&NR-U6-shRNA yielded an 8208-bp fragment, and colony PCR and sequencing analysis confirmed successful construction of the lentiviral vector shRNA-PAX6. Infection of the cells with shRNA-PAX6 caused a significant reduction of the expressions of PAX6 mRNA and protein (P<0.05) and resulted in obviously increased proliferation of U251 cells (P<0.05). Conclusion We successfully constructed the recombinant vector shRNA-PAX6 for silencing PAX6 gene. PAX6 gene silencing results in increased proliferation of U251 cells in vitro.
Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2018-01-25 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To construct a lentiviral vector for delivering short hairpin RNA(shRNA) targeting PAX6 and investigate its effect on the proliferation of glioma U251 cells in vitro. Methods Two small interfering RNAsequences targeting PAX6 gene were designed based on the reported sequence of PAX6 and annealed to form a double-stranded chain, which was inserted into a lentiviral vector to construct the recombinant lentiviral vector shRNA-PAX6. The recombinant vector was infected into U251 cells, and the expression of PAX6 mRNA and protein in the cells was detected by real-time PCR and Western blotting, respectively. The changes in the proliferation of U251 cells after the infection was assessed using MTT assay. Results Double enzyme digestion of the lentiviral vector pLKD-CMV-G&NR-U6-shRNA yielded an 8208-bp fragment, and colony PCR and sequencing analysis confirmed successful construction of the lentiviral vector shRNA-PAX6. Infection of the cells with shRNA-PAX6 caused a significant reduction of the expressions of PAX6 mRNAand protein (P<0.05) and resulted in obviously increased proliferation of U251 cells (P<0.05). Conclusion We successfully constructed the recombinant vector shRNA-PAX6 forsilencingPAX6gene.PAX6genesilencingresultsinincreasedproliferationofU251cellsinvitro.
Hits
Hits
Downloads
Comment