Isolation of single miRNA-expressing cells from zebrafish embryos

The goal of the project was to isolate single miRNA-expressing cells labelled by GFP reporter genes under the control of endogenous miRNA promoters and analyze expression levels of miRNA target genes in these cells. GFP-positive miRNA-expressing cells and GFP-negative cells from the rest of the embryos were purified at the same developmental stage to the cellular resolution using fluorescent activated cell sorting (FACS). Focus was on regulation by miR-206 and miR-133 in the developing somites and miR-124 in the developing central nervous system. Comparison of wild-type embryos and those lacking miRNAs revealed predicted targets that responded to the miRNAs and distinguished miRNA-mediated mRNA destabilization from other regulatory effects. Keywords: cell type comparison Using FACS, GFP-negative and GFP-positive cells were isolated from two different miRNA reporter wild-type lines as well as from a Dicer-mutant zebrafish line at the same developmental stage (14-16 somites). Three biological replicates for each condition were used for microarray analyses.