Advances in transgenic technology - Principles of Gene Manipulation - page 254

Box 13.1 Visible marker genes

Reporter genes are widely used for in vitro assays of
promoter activity (Box 10.1). However, reporters that
can be used as cytological or histological markers are
more versatile, because they allow gene expression
profiles to be determined in intact cells and whole
organisms.

Luciferase
CAT, GUS and b -galactosidase are stable proteins,
which persist in the cells that express them. One
problem with stable reporter proteins is that, while
they provide useful markers for gene activation, they
are less useful for assaying transcriptional repression
or rapid changes in gene activity. Luciferase was
introduced as a novel reporter gene in 1986, for
use in both plants (Ow et al. 1986) and animals
(De Wet et al. 1987). The original marker gene,
luc , was isolated from the North American firefly
Photinus pyralis and encoded a single polypeptide of
550 amino acids. The enzyme catalyses the oxidation
of luciferin, in a reaction requiring oxygen, ATP
and the presence of magnesium ions. When excess
substrate is supplied, a flash of light is emitted that is
proportional to the amount of enzyme present. This
can be detected using a luminometer, a scintillation
counter as a luminometer or even photographic film
(Wood & DeLuca 1987). Important advantages of
the luciferase system include its very high sensitivity
(more than 100-fold more sensitive than lacZ ) and
the rapid decay of light emission. Luciferase has
therefore been used to analyse the activity of genes
with oscillating expression profiles, such as the
Drosophila period gene (Brandes et al. 1996). The
amenability of the luciferase system has been
expanded by the isolation of alternative luciferases
from other organisms, which bioluminesce in different
colours (e.g. see Thompson et al. 1990). A bacterial
luciferase gene, luxA , has also been used as a marker
in transgenic plants (Koncz et al. 1987).

b -galactosidase and b -glucuronidase
The E. coli lacZ gene encodes b -galactosidase, an
enzyme that hydrolyses b -d-galactopyranosides, such
as lactose, as well as various synthetic analogues. Like
CAT, b -galactosidase activity can be assayed in vitro ,
although with the advantage that the assays are
non-radioactive. For example, cell lysates can be
assayed spectrophotometrically using the chromogenic
substrate ONPG*, which yields a soluble yellow
compound (Norton & Coffin 1985). Alternatively, a
more sensitive fluorometric assay may be preferred,
using the substrate MUG*. For histological staining,
the substrate X-gal* yields an insoluble blue
precipitate that marks cells brightly. The lacZ gene
was first expressed in mammalian cells by Hall et al.
(1983) to confirm transfection. For these experiments,
the gene was linked to the SV40 early promoter and
the mouse mammary tumour virus (MMTV) LTR
promoter. Fusions between the hsp70 promoter
and lacZ were also constructed and shown to drive
heat-shock-inducible b -galactosidase expression in
Drosophila (Lis et al. 1983). One disadvantage of
lacZ as a marker is that certain mammalian cells,
and many plants, show a high level of endogenous
b -galactosidase activity, which can obscure the
analysis of chimeric genes (Helmer et al. 1984).
The E. coli gusA gene, which encodes the enzyme
b -glucuronidase (GUS), is an acceptable alternative
(Jefferson et al. 1986). This marker is preferred in
plants, due to the minimal background activity of the
endogenous enzyme (Jefferson et al. 1987a), but has
also been used successfully in animals (e.g. Jefferson
et al. 1987b). Similar in vitro and histological assay
formats to those described for b -galactosidase are
also available for GUS, e.g. a histochemical substrate,
X-gluc*, which yields an insoluble blue precipitate.

Green fluorescent protein
The most recent addition to the growing family of
reporters is green fluorescent protein (GFP), from the
jellyfish Aequoria victoria . Over the last 5 years, this
remarkable molecule has emerged as one of the most
versatile tools in molecular and cellular biology and
is being used to investigate an increasing variety of
biological processes in bacteria, yeast, animals and
plants (reviewed by Tsien 1998, Haseloff et al.

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