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To test antimetabolites, the dU suppression tests were performed with CD1 embryos, cultured in the presence of aminopterin (1 mg/ml), 5-fluorouracil (2 mg/ml), or cycloleucine (2 mg/ml) (minimum teratogenic doses established in preliminary studies). These experiments were performed on two to four occasions, with n = 3 embryos per group.
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20
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CD1 embryos were cultured from E8.5 to E9.5 in folate-deficient serum prepared by extensive dialysis of rat serum as described (20, 26) with addition of myo-inositol (10 μg/ml). Folic acid (1.0 mg/ml) was added to the control cultures. The experiment was performed on three occasions, with n = 6 embryos per group.
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2H allele was maintained on a mixed C3H/He, 101, and CBA/Ca background. Litters from heterozygote matings were genotyped by using a Pax3-specific polymerase chain reaction (PCR) (22). Lp/+ mice were produced by mating congenic LPT/Le congenic males with inbred CBA/Ca females (Harlan Olac, UK). Litters from heterozygote matings were genotyped by Crp PCR (27). All curly tail individuals were homozygous ct/ct, of which 45 to 55% exhibited tail defects or open spinal NTDs. Embryos were categorized as affected or unaffected on the basis of posterior neuropore length at the 27-to 29-somite stage (28). The dU suppression tests were performed on mutant embryos on four to seven occasions, with n = 3 to 6 embryos per group. Animal care was in accordance with UK governmental legislation (project licence 80/00503).
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22
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Splotch and CD1 embryos were cultured from E8.5 to E10.5 in the continuous presence of folic acid (200 μg/ml), thymidine (250 μg/ml), dUMP (500 μM), or methionine (1.5 mg/ml) (maximum nonteratogenic doses established in preliminary studies).
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Pregnant splotch heterozygotes were injected intraperitoneally on E8.5 and E9.5 with folic acid or thymidine (each at 10 mg/kg body weight) or with an equivalent volume of PBS. Litters were collected at E12.5 or E13.5. We recorded the number of viable embryos and resorptions (no significant difference between groups, P > 0.05) and scored the embryos for cranial and spinal NTDs without knowledge of the treatment group. Figures of splotch embryos demonstrating prevention of NTDs by folic acid and thymidine are at: www.sciencemag.org/feature/data/981046.shl
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We thank J. Scott and D. Henderson for valuable advice and critical reading of the manuscript. Supported by the Child Health Research Appeal Trust, the Wellcome Trust, the European Union, and the National Institutes of Child Health and Human Development, USA (Cooperative Agreement HD 28882).
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