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Fig. 1 Targeting of the f8 locus produces a null allele. (A) Structure of the f8 zebrafish gene and location of the exon 4 deletion. (B) Sequencing of f8–/– cDNA demonstrates a 113 bp deletion resulting in a frameshift mutation with a premature stop codon. Amplification of (C) genomic DNA and (D) cDNA from f8+/− incrosses. (E) RT-qPCR performed in f8+/− incrosses; f8–/– fish show a significant decrease of 90% (± 28%) expression levels compared to f8+/+. The mean cycle threshold (Ct) values were compared to the reference gene β-actin to calculate the relative fold change. Error bars represent standard deviation, and statistical significance was determined by a Student t test (P = .03). (F) Survival curves of zebrafish offspring from an f8 heterozygous incross starting at 3 months of age shows a statistically significant loss of 25% of homozygotes by 1 year of age (∗∗P < .002 by log-rank Mantel-Cox testing), f8+/+ n = 23, f8+/− n = 42, f8–/– n = 29. ∗, P < .05.