ABSTRACT: In order to detect transcriptional differences between primitive and definitive hematopoietic stem and progenitor cells during regular development in the zebrafish embryo, gata1-GFP+/+(18 somites), lmo2-GFP+/+ (18 somites and 35 hpf)1 and cd41-GFP+/+ (35 hpf) cells from transgenic embryos were individually separated from GFP-/- cells by flow cytometry at the indicated stages. For each individual population, pools of 600 - 1500 transgenic embryos were collected. After RNA extraction, labelled cRNA was hybridized onto Affymetrix microarrays. Individual experiments were performed with 2 or 3 biological replicates. Experiment Overall Design: gata1-GFP+/+(18 somites), lmo2-GFP+/+ (18 somites and 35 hpf)1 and cd41-GFP+/+ (35 hpf)2 cells were separated from GFP-/- cells Experiment Overall Design: by flow cytometry at the indicated stages. Sorting was based on propidium iodide exclusion, forward scatter, and GFP Experiment Overall Design: fluorescence, using a FACSVantage flow cytometer (Beckton Dickinson). Sorted cell populations were run twice to optimize cell Experiment Overall Design: purity. Total RNA from cell-sorted populations was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy resins Experiment Overall Design: (Qiagen) according to the manufacturerâs recommendations. Total RNA was subjected to two rounds of linear amplification Experiment Overall Design: (Ambion) and hybridized to Affymetrix zebrafish Gene Chips, according to Affymetrix guidelines. After staining with a Experiment Overall Design: streptavidin-phycoerythrin conjugate (Molecular Probes), the fluorescence of bound RNA was quantitated by using a Gene Chip Experiment Overall Design: scanner (Affymetrix). The raw expression data were calculated and, after pairing of GFP+/+ and GFP-/- samples, statistically Experiment Overall Design: analyzed using methods implemented in Bioconductorâs âaffyâ package and available custom scripts 3,4. Experiment Overall Design: references: Experiment Overall Design: 1. Zhu, H. et al. Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Dev Biol 281, Experiment Overall Design: 256-69 (2005). Experiment Overall Design: 2. Lin, H. F. et al. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 106, 3803-10 (2005). Experiment Overall Design: 3. Choe, S. E., Boutros, M., Michelson, A. M., Church, G. M. & Halfon, M. S. Preferred analysis methods for Affymetrix Experiment Overall Design: GeneChips revealed by a wholly defined control dataset. Genome Biol 6, R16 (2005). Experiment Overall Design: 4. Weber, G. J. et al. Mutant-specific gene programs in the zebrafish. Blood 106, 521-30 (2005).
