Microarray methods provide a powerful tool for rapid quantitation of changes in global gene expression within eukaryotic cells. However, these methods suffer from the fact that most complex tissues exhibit substantial cell heterogeneity; signals from minor subpopulations of cells are lost within the general background of signals from the remaining cells. Moreover, changes in gene expression in one cell type may be offset by reciprocal changes in another. To obviate this problem, we developed methods for targeting protein fluorophores (FP's) based on gene promoters that are unique to specific cell types. Three problems were experienced with the constructs: 1) Autofluorescence (at all wavelengths) is high making FP's expressed in the cytosol difficult to visualize within lintact tissue. 2) Substantial manipulation of the tissue including incubation with enzymes at 37 degrees C is required to disperse the cells, and 3) The promoter activities are generally weak and turnover of the FP rapid such that expression per cell is low. We propose to develop novel constructs for targeting FP's to nuclei using cell type specific promoters. The expressed FP also will harbor a histone binding site to allow accumulation and stabilization of the FP in the nucleus. Our goal is to rapidly remove genetic material from a cell type of interest by tissue homogenization and isolation of fluorescent nuclei via cell sorting. The nuclei then will be used to isolate RNA. As part of this goal, we will evaluate if nuclear RNA is a good indicator a total cell RNA, and develop procedures for rapid isolation of nuclei from relevant mouse tissue for analysis of cell specific gene expression. The cells we have selected for analysis are nutrient sensing cells involved in metabolic homeostasis, and endothelial cells involved in blood pressure regulation and angiogenesis. The constructs can be used by investigators who need to identify the unique cells within mixed cell cultures. Moreover, the constructs will be used to develop mouse lines from which investigators can perform the cell specific gene array studies, as well a studies of cell function within sections of living tissue.
| Barthelson, Roger A; Lambert, Georgina M; Vanier, Cheryl et al. (2007) Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC Genomics 8:340 |
