Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our laboratory has been using protein engineering to study the relationship of primary structure to fibrinogen function. In order to examine genetically altered domains in the context of the intact, functional fibrinogen molecule, we have expressed recombinant human fibrinogen in Chinese Hamster Ovary (CHO) cells. The cDNA for each fibrinogen chain was individually cloned into the same expression vector. Each vector was cotransfected with the selection vector pRSVneo into CHO cells. In addition, the plasmids encoding A alpha and gamma were cotransfected with pRSVneo. Cells resistant to G418, a neomycin analogue, were isolated and clonal lines developed. Analysis of these lines demonstrated that CHO cells express and secrete free gamma chain, and an A alpha-gamma complex. To obtain recombinant fibrinogen, the A alpha-gamma G418-resistant clones were transfected with the B beta expression plasmid and a second selection vector, pMSVhis. Colonies resistant to neomycin and histidinol were selected and clonal lines obtained. These clones secreted biologically active recombinant human fibrinogen, which was purified from serum-free culture media by protamine-Sepharose chromatography. Analysis of the purified protein on SDS-polyacrylamide gels demonstrated a pattern indistinguishable from plasma fibrinogen. Removal of Asn-linked carbohydrate with glycosidase F revealed the presence of carbohydrate on the B beta and gamma chains, as is seen for plasma fibrinogen.
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PMID:Purification and characterization of recombinant human fibrinogen. 845 52

Despite the proven utility of green fluorescent protein (GFP) as a reporter molecule for transient gene expression, the adequacy of this marker for models requiring durable, high-level gene expression has not been fully tested. To address this issue, we performed the transfection of Chinese Hamster Ovary (CHO) cells with plasmid DNA encoding both GFP and neomycin phosphotransferase (neo) cassettes. The expression of GFP was measured after the cells were cultured in the presence or absence of G418-mediated selective pressure. After removal of G418 from the 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 possible without continued selection in G418. One cloned cell line, C463, maintained high-level green fluorescence for 18 weeks in G418 and an additional 12 weeks in nonselective medium. Our data suggest expression of GFP does not confer a growth disadvantage in mammalian cells.
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PMID:Long-term, stable expression of green fluorescent protein in mammalian cells. 924 Apr 38