Biomedical Engineering Reference
In-Depth Information
and least expensive method for acquiring gene expression data from cells. In
this system the use of only a luciferase is enough to measure gene expression.
For that, the promoter sequences of different genes are generally fused to
the sea pansy
R. reniformis
luciferase and the transcriptional fusions are
assayed for the light emission in response to different conditions (
Fig. 2)
.
This system has important advantages: fi rst, the simplicity of the
Renilla
luciferase chemistry has enabled the quantitation of
Renilla
luciferase from
living cells,
in vitro
or
in vivo
. The
in vivo
RLuc activity can be measured in
intact cells.
Renilla
luciferase requires only oxygen and coelenterazine to
generate luminescence, providing a simple luciferase system with which to
measure luminescence from living cells. However, this activity is at least 60-
fold less than the activity from cell lysates (protein extracts), suggesting that
in vivo
measurements are less effi cient than
in vitro
measurements (Srikantha
et al. 1996). For
in vivo
assays, the fungal cells are washed, resuspended in a
specifi c buffer (RLuc buffer) and mixed with a determinate concentration of
the substrate (10 µM solution of coelenterazine). In this way, the substrate
is stable in its buffer avoiding its degradation and allowing the chemical
reaction takes place. In general, luminescence is then immediately recorded
for 30s in an integration mode by using a luminometer. By contrast, for
in vitro
assays the luminescence generated by the cell lysate is recorded
for 10s. In this case, the concentration of substrate is lower (0,5 µM) and
the measurement is more immediate and effi cient. Second and more
importantly, this system is highly sensitive as 10
6
-10
7
fungal cells (for intact
cells) and 1/1,000 dilution (for protein extracts) are enough to obtain feasible
and reliable results and luminescence measurements in the assayable range
(Srikantha et al. 1996). It is remarkable that under the regulation of
C. albicans
promoters,
RLUC
can be expressed at levels several thousand times higher
than that of the background.
Some works are remarkable in this sense. Most of them are focused in
the regulation of the drug effl ux pumps
CDR1
,
CDR2
(ABC transporters)
and
MDR1
(MFS transporters) that are genes overexpressed in order to
pump the drug to the external medium (Harry et al. 2002, Sanglard et al.
1995). Another gene of interest is
ERG11
(lanosterol 14α-demethylase) whose
overexpression or mutation is also a frequent mechanism implicated in the
phenomenon of resistance to azoles (Lamb et al. 1997).
One of most common aims in these studies, in addition to the analysis
of the response to different drugs such as oxidant agents (Harry et al. 2005)
and after prolonged growth times with antifungal drugs (Song et al. 2004), is
to defi ne the promoter regions involved in the mechanisms of resistance. For
instance, elements within the
ERG11
promoter that mediate the resistance to
azoles (Oliver et al. 2007), identifi cation of negative regulatory elements that
regulates the basal transcription of
CDR1
(Gaur et al. 2005) or identifi cation
