Holger Erfle

Name : Holger Erfle

Room : 1D4.53C

Phone : (0)6221 387 8936

E-mail : erfle@embl.de

Webpage : n/a

Research Interests : High content cell biology.

Techniques : Fluorescence microscopy, cell-based assay development, DNA and cell microarray technology, DNA sequencing.

Current Project(s) :

In order to increase the screening throughput and reproducibility of microscopic experiments we have established and further improved the microarray-based expression system introduced by Ziauddin J and Sabatini DM {(2001) Microarrays of cells expressing defined cDNAs. Nature 411, 107-110}.

The method uses “reverse transfection” as the order of addition of DNA and adherent cells is reversed. In order to be able to apply reverse transfection to experiments in living cells, we decided to use chambered coverglass tissue culture dishes (Nalge Nunc International) for spotting the cDNA and siRNA molecules. These have the advantage that they easily permit the cellular medium to be changed, facilitate automation of immunostaining protocols, and most importantly allow high-resolution functional assays in living cells on any inverted microscope. For array production we developed a protocol in which the transfection reagent used is directly included with a gelatin / siRNA (DNA) / fibronectin mixture for the spotting procedure (Erfle et al., 2004).

For a spot diameter of 400 µm and a spot to spot distance of 1125 µm one can apply 384 samples per Lab-Tek chamber.

Figure 1: Workflow of “Reverse Transfection” and automatic image acquisition. The different solutions of transfection reagent, DNA/siRNA and gelatin are prepared in microwells and spotted in arrays into chambered coverglass tissue culture dishes by a spotting robot. Cultured cells are added to the chamber slides and transfected with the DNA/siRNA on the arrayed spots. In the case of ectopic gene expression 10-20 hours after transfection the fluorescence signal of the expressed, GFP labelled protein can be visualized. On the automatic scanning platform a motorized stage and motorized z-stepper perform the scanning of the live cells.

In collaboration with the groups of Jan Ellenberg (EMBL) and Roland Eils (DKFZ, Heidelberg) we have started to explore the possibilities of using this method to screen entire libraries of GFP tagged cDNAs in order to identify and cluster novel proteins which localize to specific cellular compartments in living cells (Conrad et al., 2004).

In collaboration with Philippe Bastiaens’ group we use this expression method to screen for proteins, which become post-translationally modified upon different extra-cellular stimuli such as growth factors.


	Figure 2: Spot region of an automatic “reverse” transfection. Transfection solution prepared on the Hamilton “Star” liquid handling robot, spotted with the “ChipWriter” Compact from BioRad using siRNA directed against Polo Like Kinase (Mitotic Index 51.6% (5% wild type)).

The technique we developed here is now used in the MitoCheck project (http://www.mitocheck.org). The MitoCheck Project Group at the EMBL in Heidelberg, which is one of the three major sites of the Integrated Project MitoCheck “Regulation of Mitosis by Phosphorylation - A Combined Functional Genomics, Proteomics and Chemical Biology Approach” funded by the European Commission in the Sixth Framework Programme, will carry out genome-wide loss of function screens using RNA interference and automated microscopy of human cultured cells to identify all human genes required for mitosis.

High-content screening microscopy identifies novel proteins with a putative role in secretory membrane traffic. Starkuviene, V., Liebel, U., Simpson, J.C., Erfle, H., Poustka, A., Wiemann, S. & Pepperkok, R. Genome Res 2004 Oct;14(10A):1948-56. PubMed

siRNA cell arrays for high-content screening microscopy. Erfle, H., Simpson, J.C., Bastiaens, P.I. & Pepperkok, R. Biotechniques 2004 Sep;37(3):454-8, 460, 462. PubMed

Automatic identification of subcellular phenotypes on human cell arrays. Conrad, C., Erfle, H., Warnat, P., Daigle, N., Lorch, T., Ellenberg, J., Pepperkok, R. & Eils, R. Genome Res 2004 Jun;14(6):1130-6. PubMed

A microscope-based screening platform for large-scale functional protein analysis in intact cells. Liebel, U., Starkuviene, V., Erfle, H., Simpson, J.C., Poustka, A., Wiemann, S. & Pepperkok, R. FEBS Lett 2003 Nov 20;554(3):394-8. PubMed