The Subcellular Location Of Paired-Box Gene Pax9 And Its Contribution To The Cell Biology

This research was to explore the function of Paired-box gene 9 (Pax9) including the studies about the subcellular location of Pax9 and its contribution to the cell biology.The full length Pax9 cDNA was obtained from the plasmid pB!uescript-Pax9 though PCR and cloned into pEGFP-C1 vector to generate green fluorescent protein (GFP)- Pax9 fused protein eukaryotic cell expression plasmid pEGFP-Pax9 (GenBank^? accession No. DQ022572). Different species origin mammalian cells CHO and Cos7 were transient transfected with pEGFP-Pax9 and the expression of Pax9 was detected at nucleic acid level by RT-PCR and protein level by Western blot and immunohistochemistry from subcellular area to whole cell, which proved that EGFP-Pax9 fusion protein could be formed and played it roles in cell nucleus. There was no different result between different species origin cells, which show that Pax9 had a very conserved action mechanism.Cell biology of Pax9-transfected Cos7 was measured by CellTiter 96? Aqueous one solution cell proliferation assay to generate the cell growth curve and the changes of cell cycle were carried out by the Flow CytoMeter (FCM). The growth factors Bmp2, Bmp4, Fgf8 and Shh were detected before and after Pax9 transfection to find the potent singaling pathway of Pax9. The study found that Pax9 could enhance the cell growth and the percent of G2/M phase of cell cycle was increased and no positive band of Bmp2, Bmp4, Fgf8 and Shh was detected which showed that these signals were not the target genes of Pax9 and they were no matter with the cell biology changes caused by Pax9.Gene overexpression and gene knockdown were two classic methods to study the gene function. The lentivirus plasmid pLL-Pax9 containing GFP-Pax9 fusion gene, Neo resistant gene and SV40 T antigen encoding gene was constructed. The virus was packaged by transfected 4 plasmids system to Human Embryonic Kidney cell strain 293T and used to transfect Cos7 cells. After choosing the positive cells by G418, Pax9 stable expression cell clone was generated. Green fluorescence could be observed in cell nucleus and Pax9 mRNA could be detected by RT-PCR.RNA interference was used to knockdown the Pax9 expression. Three siRNA target motifs were designed according the information of Pax9 mRNA and three methods were used to generate the Pax9 siRNAs. siRNA expression cassettes (SEC) sh-12, sh-22, sh-41, plasmid mU6pro origin siRNA expression plasmids mU6-12, mU6-22, mU6-41 and lentivirus plasmid pLentilox3.7 origin siRNA expression plasmid LL-21, LL-22, LL-41 were constructed. The preliminary experiment using plasmids mU6-12, mU6-22, mU6-41 to inhibit the Pax9 expression show that the percent of positive cell number decreased 35.38%, 48.47%, 28.97% which measured by Flow CytoMeter 24hrs after the siRNA expression plasmid were transfected and the plasmid mU6pro got the best knockdown result and 72hrs after plasmid transfected, just few cell had green fluorescence.