Rapid establishment of high-producing cell lines using dicistronic vectors with glutamine synthetase as the selection marker.
Recombinant proteins are useful tools in biological research, drug development, and drug screening. Specially designed expression vectors have been developed to introduce cDNA for recombinant protein expression in mammalian cells. We have combined a dicistronic mRNA design for expression of the recombinant protein, using glutamine synthetase (GS) for selection. A soluble form of human interleukin-4 receptor alpha chain was used as the model protein. The dicistronic vectors were compared to a standard expression vector in CHO-K1 cells in parallel experiments. Our data showed that a dicistronic vector containing an internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) was superior to a conventional expression vector in both levels of protein expression and amplification efficiency. The productivity of these clones was stable without selection pressure for an extended period of time. The GS selection system within a dicistronic vector design can achieve rapid and efficient gene amplification for protein production.
Pu
H
H
Berlex Biosciences, Richmond, CA, USA.
Cashion
L M
LM
Kretschmer
P J
PJ
Liu
Z
Z
D017398:Alternative Splicing; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line / Q000187:drug effects / Q000502:physiology*; D006224:Cricetinae; D004686:Encephalomyocarditis virus / Q000235:genetics; D005784:Gene Amplification; D005822:Genetic Vectors / Q000235:genetics*; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008717:Methionine Sulfoximine / Q000494:pharmacology; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D019948:Receptors, Interleukin-4 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D014162:Transfection
D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins; D008717:Methionine Sulfoximine; D005974:Glutamate-Ammonia Ligase