Development of Novel Technologies for Discovery of Patterns of Gene Regulation within Specific Cell Types

Abstract

Technological advances increase our abilities to address fundamental biological questions. In this study, three novel technologies for studies of gene regulation within specific cell types and a Gateway-adapted T-DNA binary vector series are described. The first innovation combines two powerful technologies, flow cytometry and DNA microarray, for profiling the global gene expression in the nuclei of specific cell types. It employs the strategy of labeling the nuclei of specific cell types by transgenically expressing GFP, fused with a core histone H2A (HTA6) under the control of cell type-specific promoters. The fluorescently labeled nuclei are released from transgenic plants by homogenization and purified by fluorescence-activated nucleus sorting. RNA samples are prepared from the sorted nuclei and subsequently amplified by in vitro transcription-based methods. The amplified RNA samples are then applied to DNA microarray analysis. The application of this technology is demonstrated in the study of the nuclei of phloem companion cells of Arabidopsis roots. The second technology is the characterization of nuclear DNA content of specific cell types within complex tissues. It employs the same strategy for labeling the nuclei of specific cell types. The fusion protein HTA6-GFP is incorporated into chromatin and the GFP signal is proportional to the DNA content. The DNA content of a specific cell type is determined by comparing the DNA content revealed by DAPI counterstaining through flow cytometric analysis. Using this method, we found that the endodermis of Arabidopsis roots had predominantly DNA contents of 4C or 8C, indicating endoreduplication. The third approach is the silencing of gene expression in specific cell types using RNAi technology. Our experiments show that in certain backgrounds RNAi induced gene silencing in specific cell types does not spread to nearby cells. These results suggest a feasible way to abrogate gene expression in certain cell types by using RNAi technology. Along with the development of the above methods, we also created a series of Gateway-adapted T-DNA binary vectors for expressing GFP fusions, for cloning tissue specific promoters, or for producing RNAi constructs. The sequence information of nine vectors has been deposited in GenBank to facilitate their use by other laboratories.