NSF DBI 0211857: Technology
Development: Novel Techniques for Discovery of Patterns of Gene Regulation
Within Complex Eukaryotic Tissues
David W. Galbraith, University
of Arizona (email@example.com).
Julia N. Bailey-Serres,
University of California, Riverside (firstname.lastname@example.org).
Fang Cheng Gong, University of
Eugenia Zanetti, University of California, Riverside
Zhang, University of Arizona
Georgina Lambert, University of
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
D.W. (2003). Global analysis of cell type-specific gene expression. Comparative
and Functional Genomics 4:208-215.
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.
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.
R., and Bailey-Serres, J. (2005). mRNA sequence features that contribute
to translational regulation in Arabidopsis. Nucleic Acids Res.
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
D.W. (2004). The rainbow of Fluorescent Proteins. Methods in Cell
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.
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.
D.W., and Birnbaum, K. (2006). Global studies of cell type-specific gene
expression in plants. Annual Reviews of Plant Biology 57:451-475.
D.W. (2006). Microarray analyses in higher plants. OMICS: A Journal of
Integrative Biology 10:455-473.
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.
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
Vectors, Constructs and Recombinant Lines
request specific methods or biological materials, please follow this link.
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.
or comments should be addressed to:David
last updated June 26, 2008