Dynamic characterization of recombinant Chinese hamster ovary cells containing an inducible c-fos promoter GFP expression system as a biomarker.
An inducible reporter gene system for Chinese Hamster Ovary (CHO-DHFR(-)) cells has been developed and characterized with respect to its dynamic properties. The reporter gene system consists of the human c-fos promoter and variants of the green fluorescence protein (GFP), either EGFP with enhanced fluorescence or its destabilized form d2EGFP. The expression of wild-type EGFP or its destabilized form was studied in CHO-DHFR(-) cells in response to serum addition or deprivation. It was shown that serum-induced c-fos promoter mediated EGFP expression was considerably higher than expression from the human CMV promoter, a strong, constitutive promoter preferentially used for high-level expression in CHO cells. However, EGFP was less suitable for studying expression dynamics than d2EGFP due to the protein's long half-life in mammalian cells. The use of d2EGFP resulted in a significant improvement in the dynamic characteristics of the biomarker, particularly when the recombinant cells were selected for high-level GFP expression by subcloning or fluorescence activated cell/sorting (FACS). GFP expression in different subclones and cell populations sorted by FACS was characterized with respect to its dynamic responses in the presence or absence of serum in the culture medium. Significant differences in the GFP expression dynamics were observed for the isolated cell populations. The experimental results indicate that cells with high-level GFP expression also have a faster dynamic response and are thus, desirable for practical application of the reporter gene system e.g. in toxicity monitoring.
Bi
J X
JX
Biochemical Engineering Division, GBF-Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Wirth
M
M
Beer
C
C
Kim
E J
EJ
Gu
M-B
MB
Zeng
A-P
AP
D000818:Animals; D015415:Biomarkers / Q000737:chemistry; D016466:CHO Cells / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism*; D002999:Clone Cells; D006224:Cricetinae; D005786:Gene Expression Regulation; D016762:Genes, fos / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008164:Luminescent Proteins / Q000097:blood / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000172:deficiency / Q000235:genetics; D014162:Transfection
D015415:Biomarkers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013762:Tetrahydrofolate Dehydrogenase