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Construction and Screening of siRNA Recombinant Plasmids for Mouse MMP-9 Gene Silencing
Tang Zhaoyong1#, Liu Yang2#, Liu Longxing1, Fang Liaoqiong1*
1College of Biotechnology, Southwestern University, Chongqing 400716, China; 2Center for Disease Control and Prevention, Lanzhou Military Area Command of Chinese People"s Liberation Army, Lanzhou 730000, China
Abstract: Specific siRNA interference target sites were designed according to the sequence of mouse MMP-9 gene in GenBank， and oligonucleotides were synthesized and inserted into pGCsi-U6/Neo/GFP plasmid to constitute the recombinant plasmids， the siRNA recombinant plasmids were transfected into B16 cells by PEI derivative， which was confirmed by flow cytometry and laser scanning confocal microscope observation， and the interfering efficiency was evaluated by Real-time quantitative RT-PCR analysis. The results showed that three MMP-9-siRNA interference plasmids were designed and constructed successfully. Transfection efficiency of the three MMP-9-siRNA recombinant plasmids were 60.04%， 63.93% and 56.27%， respectively， and the expression of MMP-9 mRNA in B16 cells was effectively down-regulated. The MMP-9-siRNA-2 had the highest interference efficiency (63%) and persistent interference. The results suggested that MMP-9-siRNA-2 is the optimal recombinant plasmid for silencing MMP-9 gene of mouse B16 cell.
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