HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M400018-MCP200v1 3/8/770    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Redmond, T. M. Articles by Uhler, M. D. Search for Related Content PubMed PubMed Citation Articles by Redmond, T. M. Articles by Uhler, M. D. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 3:770-779, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

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

Tanya M. Redmond, Xiaomei Ren, Ginger Kubish, Stephen Atkins, Sean Low and Michael D. Uhler^,,¶,||

From the Mental Health Research Institute, Neuroscience Graduate Program, and ^¶ Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0669

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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. S. Fjeldbo, K. Misund, C.-C. Gunther, M. Langaas, T. S. Steigedal, L. Thommesen, A. Laegreid, and T. Bruland Functional studies on transfected cell microarray analysed by linear regression modelling Nucleic Acids Res., September 1, 2008; 36(15): e97 - e97. [Abstract] [Full Text] [PDF]

				E. Hodges, J. S. Redelius, W. Wu, and C. Hoog Accelerated Discovery of Novel Protein Function in Cultured Human Cells Mol. Cell. Proteomics, September 1, 2005; 4(9): 1319 - 1327. [Abstract] [Full Text] [PDF]

				D. OVCHARENKO, R. JARVIS, S. HUNICKE-SMITH, K. KELNAR, and D. BROWN High-throughput RNAi screening in vitro: From cell lines to primary cells RNA, June 1, 2005; 11(6): 985 - 993. [Abstract] [Full Text] [PDF]