Purification of specific cell populations from Drosophila tissues by magnetic bead sorting, for use in gene expression profiling

Abstract

We developed a strategy to purify a small cell population from the Drosophila ovary for the purpose of gene expression profiling (used in Wang et al, 2006b). The Drosophila ovary is comprised of ovarioles, which are strings of egg chambers at successively later stages of development. Within each egg chamber, there are 16 large germ line cells encompassed by about 650 somatic epithelial follicle cells. Of this set of epithelial cells, some cells will take on specialized fates, such as the border cells. The border cells are a cluster of 6-8 cells that detach from the epithelium and actively migrate, as part of their normal development. Once they reach their target, a second population of follicle cells, centripetal cells, initiate migration. We were interested in the gene expression profiles that distinguish the migratory border and centripetal cells from the non-motile somatic cells and the germ line cells they invade, since ample evidence suggested that changes in transcription play a major role in promoting border cell migration. As they comprise less than 1% of the cells in the developing egg chamber, the migratory population first had to be purified. While fluorescence activated cell sorting (FACS) is commonly used for this purpose, we found this approach to be inconvenient and gravely damaging to the cells. Therefore we developed a magnetic bead-based cell purification method (Figure 1A). Flies expressing the transcriptional activator GAL4 in the cells of interest were crossed to flies bearing the UAS-mCD8-GFP transgene, which leads to GAL4-dependent expression of a fusion protein (mCD8-GFP) composed of the extracellular and transmembrane domains of the mouse CD8 antigen (mCD8) fused to GFP (Lee and Luo, 1999). Ovaries were dissected from female progeny carrying both transgenes and dissociated with protease treatment. We captured the migratory cell population using commercially available anti-mCD8-antibody-coated magnetic beads and a magnetic separation column. We recovered viable cells enriched up to 100 fold for the GFP-positive population. In principle, this purification method can be generalized to other cell types in the fly or to other organisms. The protocol goes on to describe the use of RNA isolated from these cells to determine gene expression profiles by microarray analysis. We also compared the expression profiles of cells from wild-type and specific mutant strains to identify target genes of critical transcription factors required for b…