Target Discovery and Validation Reviews and Protocols

9
Production of siRNA- and cDNA-Transfected Cell Arrays
on Noncoated Chambered Coverglass for High-Content
Screening Microscopy in Living Cells
Holger Erfle and Rainer Pepperkok
Summary
In this chapter, we provide a protocol for the production of transfected cell arrays in living
mammalian cells on noncoated chambered coverglass for the systematic functional analyses of
human genes by high-content screening microscopy. This method should facilitate drug target
validation by small-interfering RNAs.
Key Words: Cell arrays; genomics; high-content screening microscopy; small-interfering
RNAs; siRNA.
1. Introduction
The information available through sequencing of several genomes together with
high-throughput techniques such as protein analysis by mass spectrometry or
expression and transcription profiling by protein or DNA microarrays has the
potential to help analyze the complexity of biological processes on a comprehensive
scale (see Chapters 2, 4, 5, Volume 1). An elegant high-throughput method
allowing parallel analysis of gene function in intact living cells has been introduced
recently by Ziauddin and Sabatini (1). In this method, plasmid DNAs or smallinterfering
RNAs (siRNAs) (2) are printed together with the appropriate transfection
reagents in a gelatin matrix at defined locations on glass slides. Overlaying
these arrays with tissue culture cells results in clusters of living cells transfected
with siRNAs or expressing the respective cDNAs at each location. In combination
with automated fluorescence scanning microscopy and image processing (3,4), this
method allows the rapid analysis of gene function on a large scale in intact cells.
From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols
Volume I, Emerging Strategies for Targets and Biomarker Discovery
Edited by: M. Sioud © Humana Press Inc., Totowa, NJ
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