Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure.
The expression of genes transfected into mammalian cells is critically dependent on both the copy number and the site of integration. This conclusion was derived from the transfection of a plasmid containing three linked genes, which guarantees the same dose of each gene in the recipient cell clones. The selection pressure imposed by the usual concentrations of the neomycin analogue G418 or puromycin alone is low and yields cell clones with widely differing transcription rates. Time-consuming screening procedures, which are generally applied to obtain high-yield cell lines, can be obviated by the simultaneous transfer of two drug-resistance genes (neo plus dhfr, or neo plus pac). By a combined selection procedure only cells with high-level expression for the first marker will survive upon exposure to increasing concentrations of the second antibiotic. Generally, these cells also exhibit high expression levels for the non-selected gene. Many of the selected clones exhibit increased copy numbers of the respective gene but some of them owe their high productivity to a favorable position of only one or a few copies of the gene. Screening for high-yield clones using the combined selection protocol introduced here provides rapid and simple access to authentic and mutant gene products.
Wirth
M
M
Cell Biology and Genetics Section, GBF, Braunschweig, F.R.G.
Bode
J
J
Zettlmeissl
G
G
Hauser
H
H
D000818:Animals; D000990:Antithrombin III / Q000235:genetics; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002999:Clone Cells; D004274:DNA, Recombinant / Q000378:metabolism*; D005796:Genes; D005821:Genetic Techniques; D010957:Plasmids; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
D004274:DNA, Recombinant; D000990:Antithrombin III; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase