DEVELOPMENT OF A BICISTRONIC VECTOR DRIVEN BY THE HUMAN POLYPEPTIDE-CHAIN ELONGATION-FACTOR 1-ALPHA PROMOTER FOR CREATION OF STABLE MAMMALIAN-CELL LINES THAT EXPRESS VERY HIGH-LEVELS OF RECOMBINANT PROTEINS

We have developed a new vector for the stable expres sion of recombina nt protein in mammalian cells. in this vector, designated pEFIRES-P, b oth the recombinant cDNA and the puromycin resistant gene (pac) are tr anscribed as a single message driven by the strong human polypeptide c hain elongation factor Icu promoter. The presence of an internal ribos ome entry site ensures any clones that are resistant to puromycin also express high levels of recombinant protein encoded by cDNA cloned ups tream from the pac gene. We report the use of this vector to generate stable cell lines expressing human pyruvate carboxylase and show that following transfection and clonal selection, all twenty colonies that conferred high dose resistance to puromycin also expressed very high l evels of functional pyruvate carboxylase. Use of pEFIRES-P should perm it the rapid and efficient generation of stable cell lines for the cha racterisation of recombinant proteins. (C) 1998 Academic Press.