High content screening
From Liebel-lab
Custom designed siRNA-Screen to identify Ca activated Cl channels (Semir Jeridi)
We use RNAi in an automated Screening system to identify the gene that encodes for an elusive yet very important ion channel protein. This channel protein plays an important role in the excitation of sensory neurons (e.g. pain, olfaction) and smooth muscle cells, in development, the regulation of vascular tone, epithelial fluid secretion and it has also shown to be upregulated in several cancers. Upon activation by free Ca2+ it can conduct Cl ions in either direction over the plasma membrane, depending on the electrochemical gradient.
We use a cell line derived from precursors of rat olfactory neurons to perform RNAi experiments. This cell line functionally expresses the channel protein. A list of candidate proteins to be tested in knock-down experiments was derived from a proteome analysis of the cilia of olfactory neurons. In the cilia of these neurons the channel is abundant in high concentration.
As the knock-down of the channel in our cell line should lead to a loss of the endogenous channel function, we probe for a reduction of Ca2+ dependant Cl- fluxes upon siRNA transfection. Therefore we have developed a fluorescence based assay on these cells that uses Cl-sensitive YFP as a reporter and implemented it in an automated screening system.
This System we developed uses the fully automated Olympus IX81 fluorescence microscope and combines it with several liquid handling devices, integrated and controlled by a custom designed software. It thus allows the automated addition of several agents and simultaneous measurement of fluorescence with a (sub)cellular resolution. With this method the measurement of thousands of cells per experiment is no problem, providing a reliable statistical basis.
The first candidates resulting from this screening approach are now being further investigated using the precise but extremely time and labour intensive patch clamp technique. This project is a collaboration with the group of Prof. Stephan Frings at the Department of Molecular Physiology (University of Heidelberg).
