NSF DBI 0211857: Technology Development: Novel Techniques for Discovery of Patterns of Gene Regulation Within Complex Eukaryotic Tissues


David W. Galbraith, University of Arizona (

Julia N. Bailey-Serres, University of California, Riverside (


Project Personnel:

Postdoctoral Associates

Fang Cheng Gong, University of Arizona
Eugenia Zanetti, University of California, Riverside

Graduate Students

Chanqing Zhang, University of Arizona

Technical Support

Georgina Lambert, University of Arizona


Project Overview: 

This project will develop novel techniques for measurement of global gene expression within eukaryotic organisms which will permit analysis of the individual contributions of the different cell types contained within complex tissues.

The project comprises a series of proof-of-concept experiments primarily using Arabidopsis thaliana. Two strategies will be employed. The first is to label nuclei in a cell type-specific manner using the Green Fluorescent Protein of Aequorea victoria translationally fused to proteins that accumulate within the nucleus. This will be done by producing transgenic plants in which the production of a nuclear targeted form of GFP is under the control of cell type specific promoters. The presence of specifically labeled nuclei will be verified by fluorescence microscopy, and the individual fluorescent nuclei purified from cellular homogenates using fluorescence activated sorting. RNA will then be extracted from the nuclei and will be hybridized to DNA microarrays. Proof-of-concept will first involve validation of the different technical steps of the strategy, using promoters that are constitutively active, and then will involve analysis of global gene expression within the cell types defined using promoters of known cellular specificities. The second strategy is to label polyribosomes in an analogous cell-type specific manner. This will be done through epitope tagging of individual ribosomal proteins whose expression is regulated by promoters having cell type-specific patterns of expression. Polyribosomes will then be prepared and the associated mRNA employed for microarray hybridization. Proof-of-concept experiments, as before, will involve validation of the technical aspects of this strategy with constitutive promoters, followed by use of promoters having defined cellular specificities to chart global gene expression patterns within these cells.

The expected outcomes of this project are three-fold: a molecular toolkit (reagents, recombinant DNA molecules, plant lines), the associated descriptions of how to use this toolkit for examination of gene expression within living organisms, and the results of the experiments that are done to validate the methodology. All outcomes will be freely disseminated to the scientific community in a timely manner. The molecular toolkits will be provided to interested individuals on request. The descriptions of the methods and the results from the experiments will be posted to the project website and, as appropriate, will be published in the scientific literature. The methods are designed to be entirely general in scope, and should be transferable to other eukaryotic organisms, including those of other than the plant kingdom.



  • Galbraith, D.W. (2003). Global analysis of cell type-specific gene expression. Comparative and Functional Genomics 4:208-215.
  • Birnbaum, K., Shasha, D.E.,  Wang, J.Y., Jung, J.W., Lambert, G.M., Galbraith, D.W., and Benfey, P.N. (2003). A gene expression map of the Arabidopsis root. Science 302:1956-1960.
  • Galbraith, D.W., Elumalai, R., and Gong, F.-C. (2004).  Integrative flow cytometric and microarray approaches for use in transcriptional profiling.  Methods in Molecular Biology 263:259-279.
  • Kawaguchi, R., and Bailey-Serres, J. (2005). mRNA sequence features that contribute to translational regulation in Arabidopsis. Nucleic Acids Res. 33:955-965.
  • Kawaguchi, R., Girke, T., Bray, E.A., and Bailey-Serres, J. (2004). Differential mRNA translation contributes to gene regulation under non-stress and dehydration stress conditions in Arabidopsis thaliana. Plant Journal 38:823-839.
  • Galbraith, D.W. (2004). The rainbow of Fluorescent Proteins. Methods in Cell Biology 75:153-169.
  • Galbraith, D.W., Bartos, J., and Dolezel, J. (2004). Flow cytometry and cell sorting in plant biotechnology. In: Flow Cytometry in Biotechnology (L. Sklar, ed.), Oxford University Press, pp. 291-322.
  • Zanetti, M.E., Chang, I.-F., Gong, F.-C., Galbraith, D.W., and Bailey-Serres, J. (2005).  Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiology  138:624-635.
  • Galbraith, D.W., and Birnbaum, K. (2006). Global studies of cell type-specific gene expression in plants. Annual Reviews of Plant Biology 57:451-475.
  • Galbraith, D.W. (2006). Microarray analyses in higher plants. OMICS: A Journal of Integrative Biology 10:455-473.
  • Galbraith, D.W. (2007). Analysis of plant gene expression using flow cytometry and sorting. In: Dolezel, J., Greilhuber, J., Suda, J. (eds.): Flow Cytometry with Plant Cells, Wiley-VCH, pp.405-422.
  • Zhang, C.Q., Barthelson, R.A., Lambert, G.M., and Galbraith, D.W. (2008). Characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiology 147:30-40.

Vectors, Constructs and Recombinant Lines

Request Form:

  • To request specific methods or biological materials, please follow this link.


Acknowledgement and Disclaimer:

This material is based upon work supported by the National Science Foundation under Grant No. 0211857. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Questions or comments should be addressed to:David Galbraith
Page last updated June 26, 2008