The development of beta-lactamase as a highly versatile genetic reporter for eukaryotic cells.
We describe in this report that TEM-1 beta-lactamase has several desirable characteristics as a genetic reporter. First, it has no endogenous counterpart in eukaryotic cells and therefore provides a background-free measure of gene expression. Second, because of the uniqueness of the substrate cleavage reaction, a wide variety of substrates which are efficiently cleaved can be synthesized for beta-lactamase. Third, since the assays involve no more than addition of substrate to media, it is possible to continuously monitor a culture without destruction of the cells. Fourth, the enzyme is extremely versatile in that it can be fused to other proteins and retain activity. To demonstrate the versatility of beta-lactamase, we created three forms of the enzyme including secreted, intracellular, and membrane-bound forms of the enzyme, each form having distinct advantages as a reporter system. We also showed that levels of secreted beta-lactamase were proportional to both the levels of transfected DNA, beta-lactamase mRNA, as well as activity of the chloramphenicol acetyl transferase gene controlled by the same promoter, validating the reliability of this reporter. beta-Lactamase thus represents a novel and highly versatile genetic reporter.
Moore
J T
JT
Glaxo Wellcome, Division of Biological Sciences, Research Triangle Park, North Carolina 27709, USA.
Davis
S T
ST
Dev
I K
IK
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D017930:Genes, Reporter; D005821:Genetic Techniques; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology; D008566:Membranes / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; D001618:beta-Lactamases