1 [10]. Total RNA was extracted using ISOGEN (Nippon Gene, Japan) according to the manufacturer’s instructions, and treated with DNase using a Turbo DNA-free Kit (Ambion, TX) as prescribed by manufacturer. The quantity and the purity (A260/A280>1.6) of the total RNA were assessed using a spectrophotometer (NanoDrop ND-1000S, NanoDrop Technologies, DE). Total RNA was reverse-transcribed using H Minus M-MuLV Reverse Transcriptase (MBI Fermentas, Lithuania) primed with an oligo-dT17 primer and PCR was performed using Blend Taq®-Plus-DNA polymerase (TOYOBO, Japan) according to the manufacturer’s instructions. The sequences of the primers used in this

study are shown in Table 1. The PCR conditions consisted of an initial denaturation step of Trichostatin A research buy 94 °C for 2 min, followed by 30–45 cycles of 94 °C for 30 s, 60 °C for 30 s and a final elongation step of 72 °C for 30 s. To characterize the dolphin BMMC, the expression profiles of the hematopoietic marker genes expressed in human and/or mouse hematopoietic cells were compared with the expression profiles of PMN and PBMC by RT-PCR (Fig. 1). Hematopoietic Alectinib supplier marker

genes, such as CD34, GATA2, SCL/tal-1, NE, EPOR, GATA1 and Pax5, were strongly expressed in BMMC, but not in PMN. Slight expression of CD34, GATA2, GATA1 and Pax5 was observed in PBMC. After 14 days of culture in petridishes, at least three types

of BMMC colonies could be identified based on morphology (Fig. 2A–C). These colonies were referred to as “Type 1”, “Type 2” and “Type 3” colonies. The total colony counts per petridish were 30–40 colonies, of which each of the colonies accounted for approximately 5–20%, 30–35% and 40–60%. On the other hand, culture of the peripheral blood leukocytes under the same conditions revealed that no PMN cultures were produced and that only 0–2 colonies of Type 3 colony cells were generated from PBMC. Type 1 colonies were composed of numerous neutrophil-like cells and monocyte/macrophage-like cells, as Sinomenine well as a few megakaryocyte-like cells and eosinophil-like cells (Fig. 2D). Type 2 and 3 colonies primarily contained neutrophil-like cells with a few monocyte/macrophage-like cells and eosinophil-like cells (Fig. 2E and F). Hematopoietic marker gene expression profiles for the cells in each of the colony types are shown in Fig. 3. For three types of colonies, the hematopoietic marker gene expression profiles were compared with the morphological characters observed in the CFU assay; neutrophil markers (NE, G-CSFR and MPO), eosinophil marker (Epx), monocyte/macrophage and neutrophil marker (CD11b), monocyte/macrophage markers (M-CSFR, CD14 and MSR) were identified, but the erythrocyte markers (β-globin and EPOR) and B-cell markers (Pax5 and EBF) were not observed.