High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector.
We have constructed expression vectors for Chinese hamster ovary (CHO) cells that produce both selectable marker and recombinant cDNA from a single primary transcript via differential splicing. These vectors produce stable CHO cell clones that, when pooled, produce abundant amounts of secreted recombinant proteins compared with the amounts produced by conventional expression approaches that have selectable marker and the cDNA of interest under control of separate transcription units. Our vectors divert most of the transcript to product expression while linking it, at a fixed ratio, to dihydrofolate reductase (DHFR) expression to allow selection of stable transfectants. Pools of clones with increased expression of the product gene can be efficiently generated by selection in methotrexate. The high level of expression from pools allows convenient and rapid production of milligram amounts of recombinant proteins.
Lucas
B K
BK
Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080-4990, USA.
Giere
L M
LM
DeMarco
R A
RA
Shen
A
A
Chisholm
V
V
Crowley
C W
CW
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000096:biosynthesis; D015870:Gene Expression; D005803:Genes, Immunoglobulin / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007438:Introns / Q000235:genetics*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*
D000888:Antibodies, Anti-Idiotypic; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D016169:Lymphocyte Function-Associated Antigen-1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C026408:anti-IgE antibodies; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate