Ah-Young Chung, et al. MS, the most common primary demyelinating disease (Franklin, 2002). In MS, the major cause of primary demyelination is inflammatory damage to myelin and oligodendrocytes, but the reason for the remyelination failure is largely unknown. To understand the molecular mechanisms which control oligodendrocyte differentiation and myelina-tion, the RNAs expressed by oligodendrocyte lineage cells in culture have been profiled using microarray technology (Dugas et al., 2006; Cahoy et al., 2008; Chen et al., 2009). These studies have revealed distinct classes of RNAs that are differentially expressed during oligodendrocyte differentiation and have led to the identification of new factors such as Gpr17, which encodes a G protein- coupled receptor that controls the timing of oligodendrocyte dif-ferentiation (Chen et al., 2009), and myelin gene regulatory factor (Mrf ), which encodes a transcrip-tion factor necessary for expression of myelin genes (Cahoy et al., 2008). However, screening for genes differentially expressed in oligodendrocyte lineage cells using cultured oligodendrocyte lineage cells has limited potential to isolate genes responsible for oligodendrocyte differentiation and myelination because these processes are also controlled by extracellular ligands and molecules secreted from neurons, such as Jagged, PSA-NCAM, and LINGO-1 (Wang et al., 1998; Charles et al., 2000; Mi et al., 2005). Here, a microarray screening method was used to isolate genes involved in oligo- dendrocyte differentiation using oligodendrocyte lineage cells sorted from transgenic zebrafish. Oligodendrocyte lineage cells expressing enhanced green fluorescent protein (EGFP) were isolated by fluorescence activated cell sorting (FACS), and the gene expression profiles of undifferentiated OPCs and differentiated oligodendrocytes were compared. Seven genes not previously known to be involved in oligodendrocyte differentiation were identified, and their expression during oligodendrocyte de- velopment was validated. MATERIALS AND METHODS Animals Zebrafish embryos used in this study were wild-type AB line, Tg (olig2:eg fp) (Shin et al., 2003), Tg(sox10:eg fp) (Carney et al., 2006) and raised at 28.5oC in egg water or embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM NH2PO4, 0.7 mM NaHCO3, EM). Embryos were staged according to days post fertilization (dpf) and morphological criteria (Kimmel et al., 1995). Trichostatin A treatment Embryos were dechorionated and incubated in EM with histone- 86 www.enjournal.org deacetylase inhibitor Trichostatin A (Tsai et al., 2006) at a concent- ration of 100 ng/ml. Cell dissociation for flow cytometry Embryos were raised until appropriate stage and anesthetized with ethyl 3-aminobenzoate methane-sulfonic acid on ice. A thousand embryos were chopped into several pieces and transferred into 1.5 ml eppendorf tube. Collected samples were rinsed with calcium-, magnesium-free Ringers solution for 20 min, then washed with Dulbecco’s phosphate buffered saline (D-PBS) (GIBCO) three times. Tissue was digested with 100 µl of D-PBS con-taining 35 µl of Liberase Blendzyme 3, followed by incubation at 29oC for 15 min with occasional mixing. After incubation, 1 ml of trypsin (GIBCO) was added and incubated at 29oC for 15 min until complete dissociation. To inactivate trypsin, we added 2 ml of D-PBS containing 10% fetal bovine serum (FBS) and filtered the solution through a cell strainer (BD Falcon) to remove undissociated cell clusters. We collected dissociated cells by cen-trifugation at 300 g at 4oC for 5 min and rinsed twice with D-PBS containing 10% FBS. Dissociated cells were collected by centrifugation and resus-pended in PBS containing 1 mM EDTA, 25 mM HEPES (pH 7.0) and 1% FBS. Cell suspensions were separated into GFP+ and GFP- cells by BD FACSAria II (Becton Dickinson) performed at room temperature under sterile conditions. Isolated GFP+ and GFP- cells were collected in D-PBS/10% FBS pre-coated tubes with RNase inhibitor. RNA extraction Isolated cells were collected by low-speed cen-trifugation and homogenized in TRIzol solution (Invitrogen) subsequently. Total RNA extraction was performed as described previously (Peterson and Freeman, 2009). Isolated total RNA was cleaned up with RNeasy midi kit (Quiagen) and qualified as a microarray target samples using Bioanalyzer 2100 system (Agilent) and NanoDrop spectrophotometer. Hybridization of agilent gene chips 1 µg of qualified total RNA was amplified and labeled with Cy3, Cy5 dyes. Hybridization was performed with Zebrafish Agilent Gene Expression Microarray Chips (Agilent), followed by washing step. Hybridized chips were then scanned, nor-malized and analyzed. Semi-quantitative and quantitative RT-PCR cDNA was synthesized using ImProm-II reverse transcription system (Promega). The quantitative real-time PCR was performed using the Lightcycler system (Roche Applied Science) with http://dx.doi.org/10.5607/en.2011.20.2.85