Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein.
We have developed an improved vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pCIN, both the recombinant cDNA and the neomycin phosphotransferase selection marker are transcribed from a single promoter element. To facilitate translation of the second open reading frame, the encephalomyocarditis virus internal ribosome entry site has been inserted into the expression cassette immediately before the start codon of this sequence. We report the use of this vector to generate stable cell lines expressing the human 5-HT1Da serotonin receptor and show that following transfection and clonal selection, all ten cell lines characterized express similar and high levels of receptor (1.5-11.9 pmol receptor/mg protein). Use of pCIN should permit the rapid and efficient production of stable mammalian cell lines for the characterization of recombinant protein, as this vector appears to predispose all transfected cells to express such protein.
Rees
S
S
Receptor Systems Unit, Glaxo Wellcome Research and Development, Stevenage, Herts, UK. esr1353@ggr.co.uk@relay
Coote
J
J
Stables
J
J
Goodson
S
S
Harris
S
S
Lee
M G
MG
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D015870:Gene Expression; D005796:Genes / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011985:Receptors, Serotonin / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase