Separating, sorting, and profiling different cell populations from complex multicellular eukaryotic organs can be challenging. David W. Galbraith, Ph.D., who has a research program housed within the BIO5 Institute at the University of Arizona, notes that, “Prior to gene expression measurements, it is critical to separate out different cell types. The challenge has been not only to reliably purify those cells of interest from tissue, but also, at the same time, minimize disruption of normal cellular function during isolation.”
Enter fluorescence-activating sorting of nuclei. Dr. Galbraith and his team adopted a different approach: “Instead of focusing on the cell, we concentrated on the transcriptional center of the cell, the nucleus. We initially explored this strategy using the plant model, Arabidopsis. We produced transgenic plants expressing a histone2A-green fluorescent protein (GFP) fusion that selectively labeled the nucleus. Following gentle homogenization of the plant tissues, we employed flow cytometry and fluorescence-activated sorting to purify the nuclei from different cell types.”
The team simultaneously sorted GFP-positive and GFP-negative nuclei and used microarrays to determine which transcripts were most abundant in the GFP-positive nuclei. “We were able to determine the cell-type specific expression patterns of 12 genes that were selectively expressed in the phloem, the vascular tissue of plants,” says Dr. Galbraith. “Further, we were able to show that profiling mRNA within the nucleus accurately represents mRNAs in the cytoplasm.”
According to Dr. Galbraith, the team is now extending this paradigm to mammalian cells and tissues. By using a promoter-specific regulatory sequence and this particular marker targeted to the nucleus, the team is able to extend their analysis to many different types of cells.
In addition, investigators can make use of the many different colored fluorescent proteins that are becoming available. “For example, a mouse transgenic for combinations of fluorescent proteins targeted to different brain cells would lead to a more sophisticated and comprehensive understanding of microanatomy, physiology, and regulation of cell-type specific gene expression in that organ,” remarks Dr. Gailbraith. “Furthermore, there are also many ways to translate this technology to the study of human disease.”