Figure 1
Figure 1. Generation of afibrinogenemic zebrafish. Mutations were introduced in exon 2 of the zebrafish fga gene by ZFN-mediated editing. (A) The mutations in zebrafish lines described are represented. The exon-intron composition of the 2 zebrafish fga transcripts, encoding fibrinogen Aα (upper) and Aα-E (lower), are sketched at the top of the panel. Coding regions are broad, noncoding parts of the transcripts and introns are narrow. The arrow represents the transcriptional orientation. The mutated region of exon 2 is zoomed, showing the local single-letter amino acid sequence in the wild-type Aα or Aα-E above the nucleotide sequence within exon 2 for wild-type and mutant zebrafish described. The underlined nucleotides in the wild-type sequence are a BceAI recognition sequence, disrupted in the mutants described and used for genotyping. The blue letters represent the position of the designed interaction site for the ZFN used; only one DNA strand is shown for clarity. Hyphens represent deleted nucleotides. Mutation 1 is a 10-nucleotide deletion and 3-nucleotide insertion, mutation 2 a 14-nucleotide deletion, and mutation 3 a 7-nucleotide deletion. (B) Reduced (R) plasma preparations from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant fish were subjected to western blotting using antibodies to zebrafish fibrinogen Aα, Bβ, and γ chains and ceruloplasmin (CP) as a loading control for each blot. A second Aα blot was made with nonreduced samples to detect the fibrinogen hexamer (NonR). The additional high-molecular weight band in the mutation 1 wild-type (+/+) nonreduced Aα blot is thought to be a small amount of fibrin polymer that may have rapidly formed upon tail sectioning prior to immersion in heparin solution. Fibrinogen was not detected in any of the homozygous mutant fish plasma samples (−/−). The additional higher molecular weight anti-Aα reactive band in the mutation 3 +/+ and +/− samples, marked with an asterisk, is thought to be the Aα-E isoform. Its absence in mutation 1 and 2 +/+ and +/− samples is due to a polymorphism seen on the nonmutated fga allele in these lines that is predicted to truncate the Aα-E isoform (supplemental Data). (C) RT-PCR products run on agarose gels are shown for fga, fgb, fgg, and ceruloplasmin (cp) cDNA in wild-type (+/+), heterozygous (+/−), and homozygous mutant fish (−/−) liver samples for each zebrafish line. The − or + under each lane denotes PCR reactions made on cDNA synthesis reactions without (−) or with (+) reverse transcriptase.

Generation of afibrinogenemic zebrafish. Mutations were introduced in exon 2 of the zebrafish fga gene by ZFN-mediated editing. (A) The mutations in zebrafish lines described are represented. The exon-intron composition of the 2 zebrafish fga transcripts, encoding fibrinogen Aα (upper) and Aα-E (lower), are sketched at the top of the panel. Coding regions are broad, noncoding parts of the transcripts and introns are narrow. The arrow represents the transcriptional orientation. The mutated region of exon 2 is zoomed, showing the local single-letter amino acid sequence in the wild-type Aα or Aα-E above the nucleotide sequence within exon 2 for wild-type and mutant zebrafish described. The underlined nucleotides in the wild-type sequence are a BceAI recognition sequence, disrupted in the mutants described and used for genotyping. The blue letters represent the position of the designed interaction site for the ZFN used; only one DNA strand is shown for clarity. Hyphens represent deleted nucleotides. Mutation 1 is a 10-nucleotide deletion and 3-nucleotide insertion, mutation 2 a 14-nucleotide deletion, and mutation 3 a 7-nucleotide deletion. (B) Reduced (R) plasma preparations from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant fish were subjected to western blotting using antibodies to zebrafish fibrinogen Aα, Bβ, and γ chains and ceruloplasmin (CP) as a loading control for each blot. A second Aα blot was made with nonreduced samples to detect the fibrinogen hexamer (NonR). The additional high-molecular weight band in the mutation 1 wild-type (+/+) nonreduced Aα blot is thought to be a small amount of fibrin polymer that may have rapidly formed upon tail sectioning prior to immersion in heparin solution. Fibrinogen was not detected in any of the homozygous mutant fish plasma samples (−/−). The additional higher molecular weight anti-Aα reactive band in the mutation 3 +/+ and +/− samples, marked with an asterisk, is thought to be the Aα-E isoform. Its absence in mutation 1 and 2 +/+ and +/− samples is due to a polymorphism seen on the nonmutated fga allele in these lines that is predicted to truncate the Aα-E isoform (supplemental Data). (C) RT-PCR products run on agarose gels are shown for fga, fgb, fgg, and ceruloplasmin (cp) cDNA in wild-type (+/+), heterozygous (+/−), and homozygous mutant fish (−/−) liver samples for each zebrafish line. The − or + under each lane denotes PCR reactions made on cDNA synthesis reactions without (−) or with (+) reverse transcriptase.

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