Nature Methods
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Nature Methods 2, 875 - 883 (2005)
doi:10.1038/nmeth1105-875

The inside scoop—evaluating gene delivery methods

Laura Bonetta

 


Going the high-throughput way

Say you need to screen hundreds of cDNAs to search for genes that, when overexpressed, induce cell death. Or maybe you want to knock down several genes believed to function in the same pathway to better delineate the steps involved. The traditional way to transfect many cells at once is to place the cells in 96- or 384-well plates, add the DNA (or RNA) and transfection reagents, and then study the cells. Alternatively, the plates containing cells and DNA are loaded in a high-throughput electroporation instrument and processed. Given the amount of work involved, these protocols typically use robots to dispense the reagents and cells, as well as automated plate readers or microscopes to analyze the results.

In an effort to simplify high-throughput transfection protocols Ziauddin and Sabatini3 developed a new method based on microarray technology. In a transfection microarray, plasmid DNA dissolved in a gelatin solution is printed on a glass slide and then covered with a lipid-based transfection reagent. After removing the excess reagent, the slide is placed in a culture dish and covered with cells in medium. Cells growing on the printed areas take up the DNA creating spots of localized transfection within a lawn of nontransfected cells. (In an alternative version of this method the lipid-based transfection reagent is added to the DNA prior to printing.) Because cells are added to the reagent, this approach was called 'reverse transfection'.

A recent study describes another procedure for conducting parallel cell transfections on microscope coverslip arrays, but using a 'forward' methodology4. "We achieved transfection of a variety of cell lines using magnetic beads coated with PCR products," says Mark Isalan of the European Molecular Biology Laboratory and first author of the study. According to the protocol, cells are grown on a glass coverslip or slide to which magnetic microbeads coated with DNA are added. Using magnets, the beads are directed to the surface of individual cells where transfection occurs. "We can control the position of the beads to determine which cells to transfect," explains Isalan. "We can deliver DNA with micrometer resolution." The efficacy of the technique, which also works with 96-well plates, is greatly enhanced by adding a transfection reagent to the mix.

Isalan et al.5 used this technology to engineer an artificial version of a gene network involved in embryonic patterning in the fruit fly. The scientists created a synthetic embryo by using a tiny plastic chamber containing various purified genes, proteins, metabolites and cell extracts. Some of the genes were attached to magnetic microbeads, so that they could be directed to specific locations using magnets anchored to the bottom of the chamber, to form a gene expression network.

 
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