Reverse Transfection Homepage

GFP_gridThis web site describes a microarray-based system for the functional analysis in mammalian cells of many genes in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with solutions containing different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of non-transfected cells. We call this approach 'reverse transfection' and we have developed two methods to reverse transfect cells. The image to the right is an example of a cell microarray made via reverse transfection. It is a laser scan image of a 14 X 10 microarray of cell clusters expressing Green Fluorescent Protein (GFP). It was made by transfecting a slide spotted with a mammalian expression vector containing the cDNA for GFP. Each cluster is about 120-150 µm in diameter and contains 50-80 transfected cells.

By printing sets of complementary DNAs (cDNAs) cloned in expression vectors, we can make microarrays whose features are groups of live cells that express a defined cDNA at each location. These 'transfected cell microarrays' should be of broad utility for the high-throughput expression cloning of genes, particularly in areas such as signal transduction and drug discovery. For many applications these arrays can serve as substitutes for protein microarrays, particularly for proteins that are difficult to purify, such as membrane proteins.

The schematic to the left illustrates the steps followed in making transfected cell microarrays using the 'gelatin-DNA' method. Plasmid DNA dissolved in an aqueous gelatin solution is printed on a glass slide using a robotic arrayer. The slide is dried and the printed array covered with a lipid-based transfection reagent. After removal of the transfection reagent, the slide is placed in a culture dish and covered with cells in medium. The transfected cell microarray forms in 1-2 days and is then ready for downstream assays. The method illustrated is the 'gelatin-DNA' method of the reverse transfection approach. We have also developed a variant called the 'lipid-DNA' method in which the lipid-based transfection reagent is added to the DNA prior to printing. This web site describes how to implement both methods. Download detailed methods here.

  


*Table of Contents *Sabatini Lab *Whitehead Institute for Biomedical Research*