Monitoring gene expression in Chinese hamster ovary cells using secreted apoaequorin.
A luminescence method for monitoring gene expression in Chinese hamster ovary cells using apoaequorin as a secreted reporter enzyme is described. In this method, the cell is not disrupted prior to assay as in the earlier aequorin procedure and in the firefly method. The apoaequorin secretion vector is constructed by fusing the DNA fragment of the signal peptide sequence of human follistatin to the apoaequorin gene. Transfection of Chinese hamster ovary cells with the vector causes the apoaequorin to be secreted directly into the culture medium. Assay is carried out by removing a small aliquot of the culture medium, incubating it with coelenterazine, and adding Ca2+ to trigger light emission from the regenerated aequorin. The light intensity is measured with a photomultiplier photometer and is proportional to the amount of apoaequorin present. The method is highly specific and sensitive and can be carried out in a relatively short period of time.
Inouye
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S
Yokohama Research Center, Chisso Corporation, Kanagawa, Japan.
Tsuji
F I
FI
D000331:Aequorin / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA; D015870:Gene Expression; D005821:Genetic Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D014162:Transfection
D001059:Apoproteins; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin; D004247:DNA