Technique / Molecular Biology / Transfection and transduction / Reporter gene assay

Reporter gene assay
(Curr Protoc Mol Biol.)
1: Curr Protoc Mol Biol. 2001 May;Chapter 9:Unit9.7A.

Isotopic assays for reporter gene activity.

Kingston RE, Sheen J, Moore D.

Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts,
USA.

This unit describes two widely used reporter systems that are based on
radioactive detection assays. The first assay uses chloramphenicol
acetyltransferase (CAT) activity as a measure of the level of expression of a
transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group
from acetyl CoA (or any of several other acyl CoA cofactors) to chloramphenicol.
In the assays described here, transfected cells are harvested and lysed, and then
acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and
modified derivatives of the antibiotic are separated from the starting material
using either thin-layer chromatography or phase-extraction. The second reporter
system uses a kit to perform a simple two-site radioimmunoassay to quantitate the
amount of human growth hormone (hGH) secreted into culture medium by transfected
cells. Medium is incubated with 125I-labeled antibody specific for hGH, and
immune complexes are collected by an avidin-coated bead. The quantity of hormone
is determined based on comparison with a standard curve.

2: Curr Protoc Mol Biol. 2001 May;Chapter 9:Unit9.6.

Overview of genetic reporter systems.

Kain SR, Ganguly S.

Clontech Laboratories, Inc., Palo Alto, California, USA.

One method to gauge changes in transcription is to link a presumed cis-acting
sequence(s) from a gene of interest to the coding sequence for an unrelated
reporter gene. Following introduction of the chimeric reporter construct into an
appropriate cell type or animal, measurement of reporter-gene product provides an
indirect estimate of the induction in gene expression directed by the regulatory
sequences. Numerous in vitro and in vivo reporters genes are discussed in this
overview with regard to important issues regarding the selection of a
transcription reporter gene, and their applications and limitations.