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However, these first experiments have highlighted some difficulties.
Indeed, biosensor overexpression can lead to abnormal embryonic develop-
ment or lethality, depending on promoter strength, simply because the
cAMP biosensor is built on an active PKA enzyme bearing its intact catalytic
activity.
172
It is therefore strongly suggested to use biosensors that have no
biological effect, such as AKAR, and, indeed, the expression of these sensors
have proved to be harmless and did not affect the memory processes in the
living drosophila.
175
The depth at which measurements must be done, com-
bined with the required low KAR expression levels, render measuring rel-
evant variations more difficult.
174
The use of a two-photon excitation
microscope, which increases the imaging depth and reduces the phototox-
icity, must be preferred for opaque tissues or deep imaging for both ratio
imaging and TD FLIM experiments, and indeed has provided new insights
into cAMP/PKA signaling processes in living drosophila.
175
Generation of transgenic mice expressing FRET-based biosensor remains a
challenge,
176
for example, because of the two fluorophore's cDNAs recombi-
nation.
177
Although this has already been done, the low expression levels (prob-
ably due to gene silencing) complicate the imaging procedure.
178
Nevertheless,
transgenic mice were generated for some KARs including EKAR and AKAR
(with a high expression level), and successful kinase activity measurements were
realized notably in the auricular skin and small intestine.
177
Another line has
been developed with a ubiquitous cAMP sensor expression which allowed di-
rect cAMP measurement on a variety of primary cell preparations.
179
As a per-
spective, transgenic animals can be designed for ubiquitous or tissue-specific
KAR expression. Furthermore, the combination of animals expressing a
KAR and mutated/KO/overexpressing gene involved in the regulation of
the kinase of interest can increase our understanding of regulatory networks.
While somewhat challenging in a practical way, kinase activity measure-
ments, and biosensors in general, have helped scientists in better their un-
derstanding of biological processes. These tools allow the generation of
data that can be exploited by the mathematician for modeling approaches
of regulatory networks. Finally, since biosensor experiments are performed
within an intact physiological context, new light is being shed onto the spa-
tiotemporal dynamic of molecular effectors involved in the regulation cel-
lular functions.
ACKNOWLEDGMENTS
This work was encouraged and supported by two CNRS national networks: the GDR2588
and the RTmFm, as well as the University Lille1. The opportunity for such a chapter arose
thanks to the French biosensor workgroup. Financial contributions came from various
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