| The
discovery of RNA interference as a very general and versatile
tool for gene silencing has promoted great interest in the use
of this technology for deducing gene function. In support of this
development the need for relevant assays for interpreting the
biological consequences of gene silencing events has become eminent.
Redistribution®
assays are cell based assays that report on signalling events
affecting a given translocating target. Therefore Redistribution®
assays are ideally positioned to report on biological effects
of silencing known and unknown members of signalling pathways.
Below are two examples:
Silencing
of Akt isoforms – effect on FKHR translocation
The
transcription factors, FKHR, FKHRL1 and AFX, function as key regulators
of insulin signaling, cell cycle progression and apoptosis downstream
of Akt in the PI3K signalling pathway. Effect of knockdown of
three Akt isoforms was investigated by studying the effect of
FKHR translocation. Figure 1 shows the effect of silencing all
three Akt isoforms (Compare figure 1A and B). Knock down of different
combinations of Akt isoforms indicate that FKHR translocation
is primarily driven by Akt 2 and 3 in this cell line (Figure 1C).
Figure
1. Activity of Akt isoform specific siRNAs in the FKHR-EGFP Redistribution®
assay. Selective knock down of Akt isoforms has been verified
by TaqMan PCR (data not shown).
Silencing
of H-RAS – effect on Erf translocation
Effect
of H-RAS knock down in the Ras/MEK/Erk signaling pathway was investigated
using the ERF Redistribution® assay. ERF is transcriptional
repressor and member of the Ets family. Erk-dependent phosphorylation
of ERF controls its activity by modulating its subcellular localization.
Figure 2A shows efficient knock down of H-RAS by siRNA, and the
effect on ERF localization is shown in figure 2B.
Figure
2. T24 GFP-ERF1 cells were transfected with HRAS or control siRNAs.
A) Knock-down was quantified by real time RT-PCR*. B) Representative
images showing effect of HRAS siRNA in ERF1 Redistribution assay.
* Data generated through collaboration with RNAx.
This demonstrates that the effects of siRNA-mediated gene silencing
can be investigated using Redistribution® assay
technology. For more information on H-Ras knock down click
here.
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