SEIX 17-20 octobre 2005 - Atelier Calcium

stranded RNA interference (RNAi) in the study of mAChR activated calcium signalling [4].
Knockdown by RNAi of either the inositol 1,4,5-trisphosphate receptor and SERCA resulted
in a reduction, in all cells tested, of carbamylcholine-evoked calcium transients to 10% or less
of control values. This knockdown was achieved with soaking in dsRNA without the need for
transfection agents, and only
10 microgrammes of dsRNA per million cells was needed. Although RNAi knockdown of
the DM1 receptor was much less effective in reducing carbamylcholine responses, we
attribute this to the high levels of DM1 expression. [Fig. 3]
Figure 3: Application of carbamylcholine (CCh, 100 M) induces an increase of
intracellular Ca 2+ concentration [Ca 2+ ] i (A). Intracellular Ca 2+ response from cells
loaded with Fura-2/AM are presented in the panels. Timing of drug application is
indicated by a horizontal bar. Application of CCh elicits an increase of [Ca 2+ ]i in
S2-DM1 cells (A) but also in S2-DM1 cells treated with DM1 dsRNA
(B). However, no response to CCh is observed when the cells are treated with either
Ins(1,4,5)P3R (C) or SERCA (D) dsRNA.
COMBINING REVERSE GENETICS WITH CALCIUM MEASUREMENTS TO
IDENTIFY NOVEL CALCIUM SIGNALLING COMPONENTS in DROSOPHILA S2
CELLS
The Drosophila genome contains approximately 14,000 genes and, of these, many remain to
have a function assigned to them. Thus, it is likely that there will be many hitherto unknown
genes that contribute to functions such as calcium signalling. Genome-wide RNAi screens
have been fruitful in finding novel genes involved in the growth and viability of Drosophila
cells [Boutros et al 2004][6]. Our own studies (Raymond et al 2004) [5] and those of Roos et
al (2005) [7] have demonstrated the potential of using RNAi in conjunction with calcium
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