| Because
of the difficulty of identifying the components of signaling
networks in mammalian cells, we are creating and using technologies
that allow us to probe gene function in a highly parallel fashion.
Our work has lead to the development of ‘cell-based microarrays’.
The features (or spots) of these microarrays consist of clusters
of mammalian cells that either over- or under-express a particular
gene product or are under the influence of a small drug-like
molecule. The features are only 100-250 microns in diameter
and, thus, on a standard microscope slide we can create arrays
containing thousands of individual cell clusters, each with
a perturbation in a different gene. |
A
cell microarray expressing GFP in all the features. Each
spot (feature) consists of a cluster of 30-80 human cells
expressing GFP and is 100-150 microns in diameter
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With this technology we can rapidly identify candidate genes
that may underlie phenotypes of interest in mammalian cells
(e.g. cell size) as well perform synthetic effect type screens.
To create cell-based microarrays we use a robot to print onto
a surface compatible with cell attachment and proliferation
nanoliters of biodegradable polymers mixed with reagents that
perturb gene function. We then culture adherent cells on the
biopolymer-containing spots. As the polymers degrade the reagents
are released, affecting, without the need of wells to sequester
the individual reagents, gene function in defined local areas
of a cell monolayer. Using this approach we have locally introduced
into mammalian cells cDNAs in expression vectors (through
a process named ‘reverse transfection’), lentiviruses,
siRNAs, and small molecules. We can examine the cells for
alterations in particular phenotypes using techniques compatible
with cells growing on a surface, such as immunofluorescence
or in situ hybridization. We have also adapting the cell microarray
concept for screening double-stranded RNAs that mediated RNAi
in Drosophila tissue culture cells.
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