The Coexpression of KiSS-1 and p27~(kip1) Genes in the Osteosarcoam Cell Line MG63

Objective To clone the metastasis suppressor gene KiSS-1 and cell cycle dependent kinase inhibitor gene p27Kip1 from human placenta tissue,construct a bicistronic expression vector to express KiSS-1 along wiht p27Kip1 via the internal ribosome entry site(IRES),and establish stable cell line expressing these two genes.Methods Total RNA was extracted from human placenta tissue.The two opening reading frames of KiSS-1 and p27Kip1 cDNA were amplified by RT-PCR,and were subcloned into shuttle vector pIRES.The plasmid construct was transfected into the osteosarcoma MG63 cells.The expressions of KiSS-1 and p27Kip1 were detected in the transfected cells by RT-PCR and western blot.Results The recombinant plasmid construct was confirmed by methods of polymerase chain reaction(PCR),restriction enzyme digestion and sequence analysis.The interested genes were detected in the transfected cells.Conclusion The successful construction of the recombinant plasmid pIRES-KiSS-1-p27Kip1 and the establishment of cell line with stable expression of these two genes will benefit the further study about the effect of these genes on osteosarcoma cells in vitro and in vivo.