Expression Of FGFR2 IIIc And Its Mutations With Lentiviral System And Preliminary Research Of These Effect On Cells

Aim:To establish the lentiviral expression systems of FGFR2â…¢c and its gain-function mutations for farther study.In addition,to explore the molecular mechanism of proliferation and/or differentiation effected by FGFR2â…¢c and its mutations in Saos-2 and L6 cells.Method:The full length DNA sequence of FGFR2â…¢c was obtained by RT-PCR from placental.As it a templet,the FGFR2â…¢c-M252 and FGFR2â…¢c-M(252+253) mutations were introduced by overlap-PCR with appropriate primers encoding the single base-pair change.All above genes were cloned into pENTA-11 to be as the entry clones.Then,the LR recombination reactions between each entry clone and pLenti 6/5V-DEST were performed.The identified recombons were co-transfect the 293FT producer cell line with PackingMix vectors to produce lentivirus by using Lipofectamine^TM 2000.Each kind viral supernatant were harvested and collected to infect Saos-2 and L6 mammalian cells.Stable cells were selected by Blasticidin for 2 weeks with regular medium changes.The individual cell clones were isolated and identified by RT-PCR at mRNA level.A series of assays were performed to study the molecular mechanism,including Real-time PCR to inspect some genes transcription level,Western blotting to measure proteins of signal pathways,MTT to evaluate cell proliferation,Alkaline phosphatase staining and Alizarin staining to bear out the grade of differentiation,and so on.Results:FGFR2â…¢c and its mutations were cloned into pLenti6/5V-DEST vector accurately and the lentivirus expressing ectogene was obtained respectively.The goal gene is integrated and expressed in each reconstructed cells.Compared with the control groups,reconstructed Saos-2 cells and L6 cells expressing FGFR2â…¢c-M252 inhibit proliferation and promote differentiation by stimulation with bFGF.Besides,reconstructed L6 cells show the p38 signal pathway be activated when bFGF was present in the medium.Conclusion:Lentiviral expression systems of FGFR2â…¢c and its mutations were cloned successfully.Saos-2 cells and L6 cells with exogenous FGFR2â…¢c and its mutations were obtained stably.All assays results suggest that FGFR2â…¢c-M252 act as a key role to stimulate Saos-2 towards to osteoblast differentiation and L6 towards to myoblast differentiation.