ZEBRA FINCH CELL LINES 281 Figure 1. PCR sexing of ZFTMA and G266 cell lines. The PCR detected female specific bands (CHD1W, ZBM1/2 repetitive sequen- ces) in control female and ZFTMA cell line. ZFTMA is female, and G266 is male. cells were diluted with DMEM and plated out in a 100-mm culture dish. The cultures were maintained at 37°C in 5% CO2 for 5–7 d, changing culture medium every day. Once the culture reached 100% confluence, the cells were passaged by harvesting with 0.05% Trypsin–1 mM EDTA Figure 2. Karyotype of ZFTMA and G266 cell lines. DAPI staining. (Invitrogen, Carlsbad, CA) and re-plating at one-third to one-fifth of dilution. To isolate immortalized cells from normal cells in the population, we repeated the passage of cells every 2–3 d. ZFTMA cell line continued to grow over 300 d (over 100 passages), and G266 cell line grew over 185 d (over 60 passages) without any sign of senescence. The cell morphologies of these two cell lines were fibroblast-like. Results and Discussion Sexes of these two cell lines were further characterized by genomic PCR using four different primer sets (Fig. 1): CHD1-Z/W (Agate et al. 2004), TGW (data not shown; Runciman et al. 1999), and ZBM1 and ZBM2 (Itoh et al. 2008). All four cases confirmed the sex of ZFTMA cells as female and of G266 cells as male (Fig. 1). The doubling time of ZFTMA cells was 9.68 h (at 300 d of culture) and of G266 cells was 8.92 h (186 d culture), which was measured by counting the cell number using hemocyto- meter. Metaphase chromosome sets were prepared according to Itoh and Arnold (2005) and stained with 4′,6-diamidino- 2-phenylindole (DAPI). Although the exact number of chromosomes has not been determined because of the difficulty in counting microchromosomes, ZFTMA cells were tetraploid and G266 cells were diploid (Fig. 2). In vitro experiments using cell culture system offer many advantages, including advantageous experimental design and speed, reduction in use of animals, and ease of manipulation of gene expression. Cell culture can be a useful source of DNA, RNA, and protein due to high rates of cell proliferation. In some cases, it is critical to use a cell line from the species that is being investigated, for example, when testing the efficiency of RNAi before applying the RNAi molecule in vivo. Hopefully these first two zebra finch cell lines will offer experimental advantages to those studying this valuable model system.