LONG-TERM, STABLE EXPRESSION OF GREEN FLUORESCENT PROTEIN IN MAMMALIAN-CELLS

Despite the proven utility of green fluorescent protein (GFP) as a rep orter molecule for transient gene expression, the adequacy of this mar ker for models requiring durable, high-level gene expression has not b een fully tested. To address this issue, we performed the transfection of Chinese Hamster Ovary (CHO) cells with plasmid DNA encoding both G FP and neomycin phosphotransferase (neo) cassettes. The expression of GFP was measured after the cells were cultured in the presence or abse nce of G418-mediated selective pressure. After removal of G418 from th e growth medium, the percentage of pooled G418 resistant transfectants which co-expressed the GFP transgene increased or remained unchanged. Flow cytometric and visual isolation of GFP-expressing cells was poss ible without continued selection in G418, One cloned cell line, C463, maintained high-level green fluorescence for 18 weeks in G418 and an a dditional 12 weeks in nonselective medium. Our data suggest expression of GFP does not confer a growth disadvantage in mammalian cells, (C) 1997 Academic Press.