Expression of Cre recombinase as a reporter of signal transduction in mammalian cells

✍ Scribed by Larry C Mattheakis; Susan E Olivan; Jennifer M Dias; Jeffrey P Northrop

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 153 KB
• Volume: 6
• Category: Article
• ISSN: 1074-5521
• DOI: 10.1016/s1074-5521(99)80130-6

✦ Synopsis

Background:

Cell-based reporter assays, which rely on a reporter gene under the control of a regulated promoter, are widely used to screen chemical libraries for novel receptor ligands. here, we describe a reporter system that is based on ligand-induced dna recombination to express the reporter gene. this system converts a transient activation of a signal transduction pathway into an amplified, constitutive and heritable expression of the reporter gene.

Results:

We constructed gene fusions of cre recombinase and mammalian promoters regulated by calcium, nuclear receptors or cyclic amp. reporter systems, comprising a cre gene fusion and a loxp/reporter gene, were used to study the kinetics and dose responses to compounds that activate or inhibit the corresponding signal transduction pathway. we compared these reporters with conventional reporter systems in which the reporter gene is under the direct control of the responsive promoter. reporter gene expression of the cre reporters was greater than that of conventional reporters and could be measured more than a week after adding the stimulus. for all pathways studied here, the dose responses of the cre reporters are nearly identical to those of conventional reporter systems.

Conclusions:

We have shown that cre recombinase can be regulated by a variety of signal transduction pathways. it should therefore be possible to use receptor ligands to induce phenotypic conversion of mammalian cells for use in a variety of applications. one such application is high-throughput screening, and we developed loxp/luciferase reporter genes that provide an amplified and sustained luminescent response.