Transfected cell array

Recently, transfected-cell arrays have emerged as a cost-effective alternative to protein arrays (see following sections for details). In the former approach, proteins are synthesized in an array format by cells. Thus, the protein purification step can be avoided, and different cell types can be used at the same time for investigation of tissue-specific effects. 

Transfected-cell array as a functional genomics tool... 

Transfected-cell arrays have been applied to several aspects of protein functional analysis. Cell array-driven protein subcellular localization studies were mentioned previously in this review. In another example, the combination of transfected-cell technology with the mammalian two-hybrid system led to a powerful tool for screening whole cDNA libraries for proteins interacting with the gene product used as bait. An advantage of this PPI screening system is that the assay is performed in mammalian cells. Thus, interactions dependent on PTMs can be detected (Fiebitz et al., submitted). 

Fig. 3 Transfected cell array (TCA). siRNA/shRNA is arrayed on a solid support and mixed with cationic lipid to enhance the transfection efficiency. Then the cells are plated and grown on the solid support. As these cells uptake the siRNA/shRNA, phenotypes corresponding to specific silencing become detectable and the corresponding gene will be identified...

At present it is not known if all H3 receptor isoforms similarly activate the wide array of signaling pathways. In transfected cells, the rat H3(413) and H3(397) isoforms both inhibit adenylate cyclase more efficiently than does the hill length rat H3 receptor isoform (445 amino acids), but the former are less efficient in activating the MAPK pathway. Much less information is available for the human H3 receptor isoforms but shorter isoforms seem to couple more efficiently to G -proteins to inhibit cAMP production in transfected cells [40],... 

Huang etal. (2002) prepared an antibody array for the simultaneous detection of 43 cytokines. They were able to verify the down-regulation of MCP-1 cytokine in transfected cells (human glioblastoma cells transfected with cx43 expression vector) relative to control cells. The antibody array is an emerging technology. In at least one study based upon the use of a commercial membrane format, the cytokine microarray failed to accurately determine cytokine levels in bacterial and lipopolysaccharide (LPS)-stimu-lated whole human blood (Copeland, 2004). 

Cell microarrays have also been fabricated. Ziauddin and Sabatini (2001) demonstrated the ability to transfect cells cultured onto plasmid DNA arrayed in gelatin on a standard DNA microarray slide. Xu (2002) printed down cells in the form of high density microarrays on permeable membranes and demonstrated phenotypic assay performance with the immobilized cells. The commercialization of viable cell arrays will permit an even closer look at cell-mediated events during the drug discovery process. 

Austen BM, Frears ER, Davies H. The use of solid ProteinChip arrays to monitor production of Alzheimer s beta amyloid in transfected cells. J Pept Sci 2000 6 459-469. 

Fig. 2 Cell arrays can be applied for screening of protein-protein interactions in mammalian cells. Expression plasmids containing cDNAs for bait, reporter, and prey proteins (A) are spotted on the glass surface (B) and reverse transfected into adhesive cell line (C). After 3 days of incubation, proteins interactions are detected by the measurement of the reporter protein fluorescence (D). Since transfected cell clusters are separated by nontransfected cells, hundreds of different prey constructs can be investigated in a single experiment...

Fig. 3 RNA interference screens using cell arrays. siRNAs are spotted using a spotting robot at a high density on a microscopic slide (A). The quality of the generated microarray can be controlled by detection of the fluorescence from rhodamine-labeled siRNA (B). Efficiency of siRNA transfection can be monitored on a single-cell level using DAPl-counter-staining of nuclei (D). (Vanhecke et at, unpublished data)...

In this chapter we describe protocols for reverse transfection to generate mammalian cell arrays for systematic gene knock-downs by RNAi or knock-ins by ectopic cDNA expression. The method is suitable for high content screening microscopy at a high spatial and temporal resolution allowing even time-lapse analysis of hundreds of samples in parallel. 

The method comprises five individual steps (see Fig. 1), including the preparation of the transfection solutions, followed by their spotting onto a cell substrate (e.g., Lab-Tek culture dishes, Nalge Nunc International, Rochester, NY), plating of the cells onto the arrays of spotted transfection solutions, preparation of the transfected cells for functional analysis, and finally the analysis of transfected cells by high content screening microscopy. 

To generate arrays of plasmid, transfect cells by calcium phosphate-DNA coprecipitation, using 1 ml of precipitate for 3 ml of cells. The precipitate contains 20 xg of supercoiled plasmid DNA/ml, a mixture of the plasmid of interest, and a plasmid carrying a selectable marker. For S2 cells, if the composition of the plasmids does not lead to selection against their presence, incorporation of approximately 1000 plasmid copies per haploid genome will result, in the form of arrays containing the same ratio of plasmids that was present in the input mix. The products of this transformation procedure in other lines cells are less completely characterized. 

Transfection techniques in vitro and ex vivo (organotypic cultures) offer an array of possibilities to investigate the consequence) s) of the introduction of foreign nucleic acids (DNA but also RNA) and or other biologically active molecules into neurons, and to combine observations with immunocytochemistry. In particular, a wide number of fluorescent reporter proteins (FRPs) can be employed for multicolor fluorescence imaging. Here we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar neuron cultures and organotypic slices based on the use of plasmid vectors and multicolor laser scanning confocal microscopy. These protocols allow analysis of live transfected cells, and, after fixation, correlative neurochemical studies. 

We may also find that Sabatini s reverse transfection method of creating "live cell" microarrays offers even greater advantage for drug discovery and development (Ziauddin and Sabatini, 2001). Their method, relying on arrayed cDNA expression vectors to transform adherent cells, provides localized real-time gene expression analysis of the putative gene product. 

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