Biomedical Engineering Reference
In-Depth Information
systems described before) by which the assay is based on combining the
chemical properties for fi refl y and
Renilla
luciferases. As described before,
these luciferases use different substrates and thus can be differentiated by
their enzymatic specifi cities. The method is based in the sequential addition
of two substrates (mixed with their respective buffers) to each sample, with
a measurement of luminescence following each addition. Addition of the
fi rst substrate (luciferin) activates the fi refl y luciferase reaction, while the
addition of the second substrate (coelenterazine) extinguishes the fi refl y
luciferase activity and initiates the
Renilla
luciferase reaction (Fig. 3). This
system is especially important when a greater density of information
is desired and the information obtained from a single-reporter assay is
insuffi cient for achieving detailed and accurate results. In this system, a
second reporter is used as a control vector and it can be used to normalize for
transfection effi ciency. Typically, the control reporter gene is engineered with
a constitutive promoter and is co transfected at the same time. Some works
are remarkable in this sense. The most common mammalian transcription
factors engineered for luciferase expression are AP-1 (Activator protein 1)
and NFMB (Nuclear factor kappa B) proteins. Recent studies in
C. neoformans
indicate that TLR2 and TLR4 have a limited contribution to the host
response to this fungal pathogen. The authors employ the HEK293 cell line
expressing either TLR2/dectin-1 or TLR4/MD2/CD14 and conclude that
NFMB is not activated in response to
C. neoformans
cells by measuring light
emission emitted by NFMB-luciferase transcriptional fusion (Nakamura et
al. 2006). By contrast and by using the same approach as stated before, other
authors show that deoxynucleic acids from
C. neoformans
and
C. albicans
activate myeloid dendritic cells via NFMB activation and, therefore, in a
TLR9 Dependent manner (Miyazato et al. 2009, Nakamura et al. 2008).
AP-1, another transcription factor, has been also assayed for light emission
using similar methods. A recent study show that
A.
fumigatus
is able to
trigger the AP-1 activation via dectin-1/Syk pathway in response to the
β-glucans exposed at the surface of the fungal pathogen (Toyotome et al.
2008). Comparable results have been recently obtained in
C. albicans
but in
this case, AP 1 activation is triggered by the presence of signal transduction
pathway mutants that control the β-glucan exposure at the cell surface
(Galan-Diez et al. 2010)
Bioluminescent Reporters as a Genetic Tool for
in vivo
Imaging
In recent years,
in vivo
imaging techniques have emerged as a powerful
tool to complement conventional assays of microbial pathogenesis
and to monitor the spatial and temporal progression of infection/gene
