Abstract:

The aim of the present study was to construct a salivary-glands-specific expression vector, which including dual screen markers gene and fibrolytic enzymes gene, and then it was transfected into porcine fetal fibroblast cells to provide fibrolytic enzymes transgenic donor cells for cloned pigs. The sequences of dual screen marker gene NEO-T2A-EGFP and fibrolytic enzyme gene SP-EGX-F2A-BGL1 were amplified by SOEPCR and PCR, respectively, and inserted into the salivary-glands-specific expression vector to generate pPSP-SP-EGX-F2A-BGL1-CMV-NEO-T2A-EGFP. After identification by PCR, restrictive enzymes digestion and sequencing, the vector was transfected into porcine fetal fibroblast cells with liposome, and the stably transfected cells were screened by G418 selection and EGFP and identified by PCR and Western blot analysis. The result showed that the salivary-glands-specific expression vector with dual screen marker genes and fibrolytic enzyme genes was constructed, and the stable transgenic porcine fetal fibroblast cell lines were established. The study paves the way for further research on production of cloned pig expressing fibrolytic enzymes in their salivary glands.