Biology Reference
In-Depth Information
2. YC3.3er
YC3.3er (the citrine-based sensor) was expressed in the beta cells of transgenic
mice under the control of the mouse Insulin 1 promoter (
Hara
et al.
, 2004
). The
sensor signal could be detected in isolated pancreatic islets and addition of thapsi-
gargin or carbachol gave the expected decrease in the 535/485 emission ratio.
3. Camgaroos and Inverse Pericam
UAS/Gal 4 expression was used to create transgenic
Drosophila
that expressed
camgaroos-1 and-2 in the mushroom bodies of adult brain (
Yu
et al.
, 2003
).
Dissected fly brains were used. Camgaroo-2 fluorescence in the mushroom bodies
was much more intense than that of camgaroo-1, but the camgaroo-1 emission
ratio signal on potassium depolarization was more than double that of camgaroo-2
(38% vs. 14% in the mushroom body lobe and 83% vs. 28% in the mushroom body
itself ). It was shown that these increases were not due to changes in pH. Applica-
tion of the putative mushroom body transmitter, acetylcholine, causes ratio
changes of a few percent. In this setting, camgaroo-2, although brighter, showed
substantially lower ratio changes than camgaroo-1; it also underwent significantly
faster photobleaching.
Inverse pericam is an intensity-coded sensor that decreases its fluorescence as
calcium increases. Addition of DsRed2 to the C-terminal of inverse pericam
produces a ratiometric indicator whose 615/510 nm emission ratio increases as
calcium increases. This indicator (DsRed2-referenced inverse pericam (DRIP))
requires dual excitation and dual emission optics (
Shimozono
et al.
, 2004
). The
DsRed2 fluorescence is a passive, calcium-independent signal that is proportional
to the concentration of the sensor and helps control for alterations in overall
fluorescence intensity due for example to movement artifacts. DRIP was expressed
transgenically in worms under the control of the myo 2 promoter that is specific for
pharyngeal muscle. Ratio changes of 30-40% were measured in worms undergoing
fast pharyngeal pumping.
After screening six sensors (flash pericam, inverse pericam, G-CaMP, camgaroo-2,
YC2.12, and YC3.12) for calcium sensitivity in stably transfected fibroblast cell lines,
the two with the greatest dynamic range (inverse pericam:
40% and camgaroo-2:
รพ
170%), together withYC3.12 that gave inconclusive results in the fibroblast expres-
sion screen but is optimized for expression at 37
C, were used to generate transgenic
mice under the control of the TET expression system (
Hasan
et al.
,2004
); the TET
system allows tissue-specific expression by crossing the TET mice with mice expres-
sing the TET transactivator under tissue-specific control. TET sensor mice were
crossed with a line expressing the transactivator under the control of the alpha-
calmodulin/calcium dependent kinase II (
a
CamKII) promoter. All mice developed
normally. Five highly expressing lines were obtained out of 36 transgenic lines: two
YC3.12, two camgaroo-2, and one inverse pericam. Expression patterns in brain
slices and excised retina were analyzed by two-photon microscopy. They appeared to
