Biomedical Engineering Reference
In-Depth Information
indicate their relevance in basic research as genetic tools to analyze and
monitor
in vitro
and
in vivo
gene expression.
Bioluminescent Genes Reporters
Bioluminescence is a natural phenomenon by which living organisms
create their own light by means of a biochemical reaction. In the last decade,
this phenomenon has been widely used in basic research, particularly in
molecular biology and biotechnology where bioluminescence enzymes
have been extensively used as reporters. Reporter genes encoding
luciferases have provided a very rapid and highly sensitive method to
analyze regulation of gene expression (Bronstein et al. 1994, Contag et al.
1995, Doyle et al. 2004, Hutchens and Luker 2007, Luker and Luker 2008,
Wick 1989). Luciferase genes have been cloned from different bacteria,
insects and other marine organisms: these include the luciferases encoded
by the bacteria
Photorhabdus luminescens
(
luxCDABE
) (Winson et al. 1998),
beetles (Firefl y,
Photinus pyralis
(FLuc); click beetle in green; Brazilian
Cratomorphus distinctus
(Emerald Luc; ELuc) and click beetle in red;
Caribbean
Pyroforus plagiophthalamus
(CBR) (Ow et al. 1986, Wood et al.
1989), the sea pansy
Renilla reniformis
(RLuc) (Srikantha et al. 1996), the
marine copepod
Gaussia princeps
(GLuc) (Verhaegent and Christopoulos
2002), the jellyfi sh;
Aequorea Victoria;
aequorin (De et al. 1996), the sea fi refl y
Vargula
and the dinofl agellate
Gonyaulax
(Maeda et al. 1996, McMurry and
Hastings 1972). Of these, luciferases from fi refl y, beetles and sea pansy
have found more general use.
Firefl y luciferase is a monomeric enzyme that catalyzes a two-step
oxidation reaction to produce light in the presence of luciferin (its substrate)
in an ATP-dependent manner. The fi rst step is the activation of the luciferyl
carboxylate by ATP to generate a reactive anhydride that, in a second step,
reacts with oxygen to produce oxyluciferin and CO
2
which is accompanied
by light emission. Click beetle luciferases belong to the same family as
fi refl y luciferases (beetle luciferases) and the mechanism of light emission
is similar. The main difference between them is the variety of luminescence
colours that beetle luciferases emit (see later in imaging section)
(Fig 1A).
Renilla and Gaussia
luciferases (also monomeric enzymes) catalyze the
oxidation of coelenterazine (their substrate) to generate coelenteramide in
an ATP and calcium independent manner (Fig 1B).
These luciferases have been used most widely in high-throughput
screening. The beetle luciferases are the most versatile of this group (Doyle
et al. 2006b). Click beetle luciferases are becoming better known and present
a range of luminescence color options which are mainly exploited in dual
color assays in imaging experiments (see later) (Hida et al. 2009).
Renilla
