Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

Program in Gene Function and Expression, University of Massachusetts Medical School, Lazare Research Building, Room 617, 364 Plantation Street, Worcester, MA 01605, USA.
Developmental Biology (Impact Factor: 3.55). 12/2006; 299(2):551-62. DOI: 10.1016/j.ydbio.2006.08.020
Source: PubMed


In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.

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Available from: Julio D Amigo, Aug 08, 2014
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    • "Embryos were prepared for FACS as described byCovassin et al. (2006). The dsRed-positive cells were sorted from flk1:GFP/gata1: dsRed transgenic embryos using MoFlo XDP (Beckman). "
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    • "After washing and counting of cells, magnetic beads (anti-PE MicroBeads, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added and allowed to bind, before washing and magnetic separation on LS Midi Columns using a MidiMACS Separator (Miltenyi Biotec). For isolation of leukocytes from Tg(mpeg1:mCherry-F) UMSF001 and Tg(mpx:egfp) i114 zebrafish, larvae at 5-6 days post fertilization (dpf) were dissociated according to Covassin et al. (2006). In short, anaesthetized embryos were dechorionated using pronase treatment , rinsed in calcium-free Ringer solution, followed by digestion with 0.25% trypsin and 1 mM EDTA. "
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