Biomedical Engineering Reference
In-Depth Information
gene and
G. princeps
luciferase (GLuc). In these studies
CTA1
-,
TRX1
- and
TTR1
/
GRX2-GFP
fusions are assayed in response to different oxidant
conditions and during systemic infection in a mouse kidney (Enjalbert et al.
2007). Similar studies have been addressed using
C. neoformans
as a model
of fungal pathogen. In this case and by means of the GFP-reporter, the
LAC1/2
-
GFP
transcriptional fusions (laccase genes) were checked in order
to verify the expression of these genes in the macrophage—
C. neoformans
model (Fan et al. 2005).
In this point, it is important to note that FPs (GFP and others) have
been traditionally used as gene reporters providing excellent results and
contributing, in a very special manner, to the scientifi c knowledge. It is
important to remark that bioluminescence have some advantages over
fl uorescence. The most basic difference is that the excited states produced in
luminescence are the product of exothermic reactions, whereas absorption
of light is the phenomenon by which the excited states are created in
fluorescence. This difference significantly affects the character of the
photometric assay. Therefore, the photons produced in the luminescence
reaction, which are not required to create the excited states, do not constitute
an inherent background when measuring photon effl ux from a sample and
permit a precise measurement of very small changes in light. By contrast,
fl uorescence-based assays tend to be much brighter, since the photon used
to create the excited states can originate background into a sample at a
very high rate. In addition, fl uorescence assays can also be limited by the
presence of interfering fl uorophores within the samples.
In addition to the sea pansy RLuc luciferase, another codon optimized
version of GLuc has been used to analyse gene expression
in vivo
. This
luciferase is also a suitable tool as a sensitive reporter for studies of gene
regulation (Enjalbert et al. 2009) (see later in imaging section).
From the cell host point of view, invasion of microbial pathogens into
tissues results in the development of infl ammatory responses, which are
initiated by recognition of Pathogen Associated Molecular Patterns (PAMPs)
via Pattern Recognition Receptors (PRRs), such as Toll Like Receptors (TLRs)
or C-type Lectin Receptors (CLRs) (Arana et al. 2009). As a consequence,
intracellular signalling pathways are triggered and are responsible for
transducing the intracellular signals that are ultimately responsible for the
functional activity of the receptor. These responses may also be quantifi ed
by using bioluminescent reporters generating genetic constructions of
gene promoters fused to a determinate luciferase. In general, a stable or
transient cell line, previously transfected to express receptors (TLRs or
CLRs), is engineered to co express a promoter region of a transcription factor
of interest fused to the fi refl y or the
Renilla
luciferase. These experiments
generally require a dual-reporter system (in contrast with the luciferase
