Microarray Transfection Analysis of Transcriptional Regulation by cAMP-dependent Protein Kinase

Abstract

A wide variety of bioinformatic tools have been described to characterize potential transcriptional regulatory mechanisms based on genomic sequence analysis and microarray hybridization studies. However, these regulatory mechanisms are still experimentally verified using transient transfection methods. Current transfection methods are limited both by their large scale and by the low level of efficiency for certain cell types. Our goals were to develop a microarray-based transfection method that could be optimized for different cell types and that would be useful in reporter assays of transcriptional regulation. Here we describe a novel transfection method, termed STEP (surface transfection and expression protocol), which employs microarray-based DNA transfection of adherent cells in the functional analysis of transcriptional regulation. In STEP, recombinant proteins with biological activities designed to enhance transfection are complexed with expression vector DNAs prior to spotting on microscope slides. The recombinant proteins used in STEP complexes can be varied to increase the efficiency for different cell types. We demonstrate that STEP efficiently transfects both supercoiled plasmids and PCR-generated linear expression cassettes. A co-transfection assay using effector expression vectors encoding the cAMP-dependent protein kinase (PKA), as well as reporter vectors containing PKA-regulated promoters, showed that STEP transfection allows detection and quantitation of transcriptional regulation by this protein kinase. Because bioinformatic studies often result in the identification of many putative regulatory elements and signaling pathways, this approach should be of utility in high-throughput functional genomic studies of transcriptional regulation.

Cell microarrays and RNA interference chip away at gene function

“…Several groups have created cell microarrays that consist of arrayed spots of plasmid vectors or linear PCR products designed to express proteins of interest printed in an aqueous gelatin mixture 5,23 . A robotic microarrayer is used to print an array of nucleic acid spots on a glass slide (Fig.…”

“…Since this work was done, a handful of overexpression cell microarray studies have been published that offer technological improvements on the basic reverse transfection principle 6,[23][24][25][26] . These studies have so far been limited to assay development or smaller screens.…”

“…These studies have so far been limited to assay development or smaller screens. For example, pilot overexpression microarrays using GFP reporters were developed for the cAMP and serum response element pathways 23,26 . Examination of the subcellular localization of tagged proteins, which can help in inferring gene function, has also been demonstrated in microarray format 24,25 .…”

High-throughput RNAi screening in vitro: From cell lines to primary cells

“…First, individual siRNAs can be transfected in a multiwell format using standard transfection methods with preplated cells. Alternatively, an array-based platform has been described wherein siRNAs are spotted on a slide, to which cells are added and transfected (Ziauddin and Sabatini 2001;Chang et al 2004;Redmond et al 2004). Transfection arrays are well suited to high-throughput RNAi screening, except for experimental variability, caused by the small number of transfected cells and the limited number of cell lines that have been reported to have transfection efficiencies high enough to be used in this application.…”

Functional genomics using high-throughput RNA interference

“…A recently developed transfected-cell array (TCA) technique is being seen as a breakthrough for high throughput functional genomics in cell biology [1][2][3].…”