Global Analysis of Cell Type-Specific Gene Expression 


 

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We have developed a novel, generally-applicable means to analyze cell type-specific gene expression within eukaryotic organisms (1,2,3). This involves transgenic expression of GFP (or other Fluorescent Proteins), targeted to the nucleus and placed under the transcriptional control of cell type-specific promoters. The nuclei are then released from the organism by gentle homogenization, are stained with DAPI and are purified by fluorescence-activated sorting. Expression profiling is then done using microarrays. We have demonstrated that nuclear transcript levels are largely concordant with those of total cellular transcripts, both in plants and animals. In terms of cell type specificity, our results are consistent with those found through sorting of protoplasts (4,5). 

Analyzing and sorting nuclei is of obvious advantage for species, organs or cells that do not readily produce protoplasts.

In order to expand this technology to the scientific community at large, we have available the DNA constructions required for expression and nuclear targeting of GFP. We also are willing to provide, on a cost-recovery basis, the processes of flow sorting and nuclear expression profiling. We envisage being sent transgenic seed, which would be germinated, the plants homogenized, the nuclei purified by fluorescence-activated sorting, and the nuclear transcripts subjected to profiling using microarrays or GeneChips following the required single-round of in vitro amplification. Alternatively, the amplified RNA would be shipped back to the originating scientist for further processing.

For further information about this service, and to request the DNA constructions, please contact me by email or telephone (520.621.9153). 


References

  1. Galbraith DW (2003). Global analysis of cell type-specific gene expression.  Comparative and Functional Genomics 4:208-215.
  2. Zhang CQ, Gong FC, Lambert GM, Galbraith DW (2005). Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant Methods 1:7 doi:10.1186/1746-4811-1-7.
  3. Zhang CQ, Lambert GM, Barthelson RA, Galbraith DW (2008). Characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiology 147:30-40.
  4. Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN (2003). A gene expression map of the Arabidopsis root. Science 302:1956-1960. 
  5. Brady SM, Orlando DA, Lee J-Y, Wang JY, Kock J, Dinneny JR, Mace D, Ohler U, Benfey PN (2007) A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318:801-806.

Questions or comments should be addressed to: David Galbraith
Page last updated July 1, 2008