Biology Reference
In-Depth Information
and absorbing issues caused in mammalian tissues
[6]
, allowing them to be
more efficiently detected outside the body of a small animal. Firefly lucif-
erase, in particular, has gained the most widespread use as, at 37
°
C, this
enzyme emits at 620 nm, the longest wavelength of a luciferase at mamma-
lian body temperatures
[7]
.
Light production from luciferases occurs through the oxidation of enzyme-
specific substrates, which are coelenterazine in marine organisms
[1,8]
and
D-luciferin in terrestrial organisms
[9,10]
. Luciferase enzymes from differ-
ent species have been given the abbreviations:
luc
(firefly),
lcf
(dinoflagel-
late) and
lux
(bacterial). The BLI reaction of
Renilla
luciferase occurs by
oxidative decarboxylation of coelenterazine in the presence of oxygen to
yield oxyluciferin, CO
2
and blue light (peak = 480 nm)
[11]
. Firefly luciferase
is ATP-dependent and requires Mg
2+
and oxygen to oxidize the substrate,
D-luciferin, to produce CO
2
, AMP, pyrophosphate, oxyluciferin and yellow-
green light (peak = 562 nm)
[12]
.
The light emitted from the luciferase reporter reaction requires extremely
sensitive cameras that can detect very low levels of visible light emitted
from within an animal
[13]
. Regular charge-coupled device (CCD) cameras
have appropriate spectral ranges for detecting biological light sources, but
are not sensitive enough to detect the faint light emitting from the internal
sources. As amplification of the optical signals from the red region is par-
ticularly difficult, the sensitivity of the detectors is increased by thinning
the CCD chip and placing it in a vacuum which is cooled to temperatures as
low as −105
°
C. These cooled CCD cameras are the most common cameras
for imaging BLI.
61
The choice of which reporter gene to use for BLI depends on what is to be
monitored, in which tissue, and the intensity and duration of signal required
[7,14]
.
Renilla
and
Gaussia
luciferases are generally considered less desir-
able for
in vivo
studies for several reasons. First, the shorter wavelengths
they produce (peak = 480 nm) do not penetrate tissues as well as light from
firefly luciferase
[15]
. Also, the substrate, coelentrazine, is prone to quick
activation and degradation through auto-oxidation. The substrate is also
expensive, has low solubility, can bind to serum proteins and is rapidly
cleared from the bloodstream
[15,16]
. However, in cases in which sequen-
tial imaging is desired with multiple luciferases, the different wavelength
and short half-life can be advantageous
[15,16]
. Another advantage of these
luciferases over the firefly luciferase is that they can be used to image cells
independent of a cell's metabolic state as
Renilla
and
Gaussia
luciferases
do not require ATP. Nevertheless, the firefly luciferase, due to its better pen-
etration and longer half-life of its substrate (D-luciferin, which is not cata-
lyzed by regular mammalian tissues), has become the BLI reporter enzyme
of choice for most studies
[15,17]
.
To effectively observe the trafficking behavior and proliferation of a given
cell population by BLI, the luciferase gene must be introduced into the cell
type of interest. This can be achieved via several methods, but the method
of choice is lentiviral-based gene transfer as it causes effective gene deliv-
ery, can infect dividing and non-dividing cells, and inserts genes into the
host genome to provide stable, high expression levels of genes in culture
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