Preliminary comparison of Paired-box gene Pax9 siRNAs' gene interference abilities

Objective: RNA interference (RNAi) was named as a gene silencing phenomenon whereby small interference double-stranded RNAs (siRNA) trigger the specific degradation of a homologous mRNA; and paired-box gene Pax9 played an important role in the tooth development. This study was to preliminary evaluate the effect of Pax9 siRNAs'gene interference abilities to further study the Pax9 function. Methods: pEGFP-Pax9 was constructed previously (GenBank accession No. DQ022572) and three siRNA target sequences were selected after screening from 100bp after ATG of Pax9 mRNA (GenBank Accession No. NM_011041.1) and blasting to eliminate the homology sequence with other genes. Primers were designed according to mouse U6 promoter (GenBank Accession No. X06980) and target sequences; PCR products were cloned into plasmid mU6pro and sequenced to generate plasmids mU6-12, mU6-22, mU6-41. RNAi effiency could be measured by the percent of cells with green fluorescence as the following steps: pEGFP-Pax9 was respectively co-transfected with mU6-12, mU6-22, mU6-41 to Cos7 cells; the percent of green fluorescence cell was measured by Flow CytoMeter (FCM) after 24hr and the transfected cell was further observed under fluorescence microscope after 72hr. Results: Sequence results showed that Pax9 RNAi plasmids mU6-12, mU6-22, mU6-41 were constructed successfully and their gene interference effiencies were 35.38%, 48.47% and 28.97% respectively at 24hrs and few cell could emit green fluorescence at 72hrs. Conclusion: mU6-12, mU6-22, mU6-41 could inhibit Pax9 expression.