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High-Resolution Banding of the Mouse Chromosomes

Human chromosomes a t metaphase show approximately 320 Q-, Gor R-bands per haploid set (Paris Conference 1971). Yunis (1981a) recently demonstrated 550 to 850 well-demarcated G-bands in prometaphase chromosomes of normal lymphocytes and leukemic cells use being made of the amethopterine synchronization method. The number of bands can be increased as many as 1,200 at late-prophase (Yunis 1981b) and 2,000 at mid-prophase (Yunis 1981c) . Similar high-resolution bands of chromosomes have been obtained from human lymphocytes treated with ethidium bromide and acridine orange, which are known to inhibit both DNA and RNA synthesis (Ikeuchi and Sasaki 1979; Matsubara and Nakagome 1982), as well as by the treatment of human lymphocytes and fibroblasts with actinomycin D, which interferes with the normal chromosome condensation (Viegas-Pequignot and Dutrillaux 1976; Yu et al., 1981). We attempted to apply these methods to several kinds of mouse cells, since there has been no report published on the high resolution banding patterns of this species. This paper reports a successful delineation of sub-bands in amethopterine-treated embryonic fibroblasts. Materials and methods. Primary cultures of fibroblasts derived from 10to 12-day-old mouse embryos were grown in Eagle's minimum essential medium supplemented with L-serine (40 mg/1), glycine (7.5 mg/l), sodium pyruvate (110 mg/1) and 20% fetal calf serum (pH 7.2 adjusted by HEPES buffer). The cultures were treated with 0.1 pg/ml amethopterine for 17 hr at 37°C. They were then released from the amethopterine block by exposure to 20 pg/ml 5-bromodeoxyuridine (BUdR) for 4.55 hr at 37°C, after washing with pre-warmed serum-free medium 3 times. The cells were exposed to 0.005 pg/ml Colcemid for the final 10 min, harvested by trypsinization, swallen in 0.075 M KC1 for 20 min at 370 C, fixed with methanol-acetic acid (3: 1) , and airdried on slides.