A High-Throughput tool for the analysis of protein-protein, protein-DNA interactions and siRNA mediated gene silencing in mammalian cells.
Accomplishment of the human and mouse genome sequencing projects resulted in identification of large number of new genes for which there is little or no functional information.
A recently developed transfected-cell array (TCA) technique is a breakthrough in that context as it opens the way for high throughput functional analysis of target genes. On TCA, expression vectors and/or siRNA molecules are printed at a high density on a glass slide along with a lipid transfection reagent. When these microarrays are covered with a layer of adherent cells, only the cells growing on top of the spots become transfected, resulting in the expression or silencing of specific proteins in spatially distinctive groups of cells. The phenotypic effects of such ‘reverse transfection’ of hundreds of genes can be detected using specific cell-based bioassays.
We use the cell arrays as a platform for high throughput analysis of protein-DNA and protein-protein interactions by combining the transfected cell array with mammalian one-hybrid and two-hybrid systems. We already demonstrated that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds.
Furthermore, the detection of protein-protein interactions on transfected cell arrays allows for the in vivo screening and selection of molecules such as small peptides or siRNA aptamers, which can inhibit these interactions by co-transfection of libraries of such small molecules in cells expressing the proteins of interest.
More interestingly, a combination of reverse transfection array technique with recently developed RNA interference technologies allows the identification of the biological function of genes. It does this by monitoring the effects following the knocked-down of these genes.
Compared to functional assays performed in micro-well format, the TCA approach requires less cells, DNA/RNA and transfection and signal development reagents per number of genes tested. Therefore, cell arrays are, at the moment, one of the most cost-effective functional tools available.