Construction and identification of mouse R-spondin 1 eukaryotic expression vector

R-spondin1 (Rspo1) has been proposed to activate and synergize with the Wnt signaling pathway. It is involved in pancreas and muscle development, bone formation, limb and gonad morphogenesis, even associated with many human diseases. In this study, we amplified the full-length Rspo1 gene from mouse pancreas using RT-PCR technology. The recombinant pMD19-T-Rspo1 vector was constructed by connecting pMD19-T vector and full-length Rspo1 gene, and then the Rspo1 full-length fragment sequenced correctly was connected to pIRES2-AcGFP1 vector after double digestion with EcoR I and BamH I. The recombinant vector pRspo1-IRES2-AcGFP1 was identified by double digestion of EcoR I and BamH I. We examined the expression of green fluorescent protein (GFP) under fluorescent microscope and the mRNA expression level of Rspo1 using RT-PCR after transfection it into HEK293 cells for 48 h. These results indicated that Rspo1 eukaryotic expression vector had been constructed successfully and identified. This would lay the foundation for further research on regulatory function of Rspo1 on Wnt signaling pathways and mechanism on proliferation and differentiation of stem cells.