Reporter genes

• CAT
• luc
• b-gal (lac Z gene)

reporter gene

A gene that encodes an easily assayed product (eg. CAT) that is coupled to the upstream sequence of another gene and transfected into cells. The reporter gene can then be used to see which factors activate response elements in the upstream region of the gene of interest.

chloramphenicol acetyltransferase

Enzyme that inactivates the antibiotic chloramphenicol by acetylation. Widely used as a reporter gene.

luciferase reporter system

Reporter genes that are based on firefly luciferase gene offer luminescent detection of reporter activity. As few biological processes emit light, this assay has a very low background.

LacZ

E. coli > E. coli gene encoding beta-galactosidase. Part of the lac operon> lacoperon.

beta-galactosidase (= b-galactosidase)

Enzyme (EC 3.2.1.23) encoded by the LacZ gene, that is widely used as a reporter gene, as a variety of coloured or fluorescent compounds can be produced from appropriate substrates (typically Xgal, that produces a blue colour). LacZ is incorporated in many plasmid vectors to allow blue-white colour selection.

blue-white colour selection

Method for identifying bacterial clones containing plasmids with inserts. Many modern vectors have their polycloning site within a part of the LacZ gene encoding beta-galactosidase, which provides a -complementation in an appropriate mutant E. coli strain. This means that a re-ligated (empty) vector will produce blue colonies when grown on plates containing IPTG and Xgal, but colonies with a substantial insert in their plasmid's polycloning site are unable to produce functional beta-galactosidase, and so produce white colonies.

IPTG (= isopropyl b-D-thiogalactoside)

Used to trigger gene expression that is under the control of gal promoter> galpromoter, particularly used in expression systems for producing protein.

Xgal (= 5-bromo-4-chloro-3-indolyl-b-D-galactoside)

Chromogenic substrate for the enzyme beta-galactosidase, yielding an intense blue colour. Very widely used in blue-white colour selection of bacteria containing recombinant plasmids, and in histochemistry of tissues/cells expressing beta-galactosidase as a reporter gene.

polycloning site (= multiple cloning site; MCS)

Region of a phage or plasmid vector that has been engineered to contain a series of restriction sites that are usually unique within the entire vector. This makes it particularly easy to insert or excise (subclone) DNA fragments.