Expression of recombinant human granulocyte colony-stimulating factor in CHO dhfr- cells: new insights into the in vitro amplification expression system.
The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.
Monaco
L
L
Biotechnology Unit, DIBIT, San Raffaele Scientific Institute, Milano, Italy. monacol@dibit.hsr.it
Tagliabue
R
R
Giovanazzi
S
S
Bragonzi
A
A
Soria
M R
MR
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D018076:DNA, Complementary; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D013762:Tetrahydrofolate Dehydrogenase