Generation of cdkn2a/b+/− zebrafish using the CRISPR/Cas9 genome editing and features of leukemic E::R;cdkn2a/b+/− fish. (a) Genomic structure of the cdkn2a/b gene. Red arrowheads mark the Cas9 cut positions in exon 2, and the sequences of the three sgRNA target sites are shown. The cdkn2a/b+/− zebrafish line with a premature stop codon after 210 nucleotides was established. (b) Phenotypic and histological characteristics of leukemic E::R;cdkn2a/b+/− zebrafish. Images depict lateral views of representative control UAS:GFP zebrafish (b(I)) and leukemic zebrafish (b(II)). The E::R;cdkn2a/b+/− leukemic zebrafish developed subcutaneous bleeding in the ventral body region (highlighted by a red frame in (b(II))). An inset provides a close-up view of the bleeding (marked with a red asterisk) (b(II)). Giemsa staining of a peripheral blood smear from the UAS:GFP fish (b(III)) revealed normal nucleated erythrocytes, while Giemsa staining of a leukemic blood smear (b(IV)) showed a significant presence of lymphoblasts. Hematoxylin and Eosin staining of sagittal paraffin sections of tissues from the UAS:GFP fish (b(V,VII)) and leukemic fish (b(VI,VIII)) revealed the presence of large deposits of lymphoblasts in the kidney marrow (b(VI)) and epidermis (b(VIII), arrows) of leukemic fish. The spleen was enlarged in the leukemic E::R;cdkn2a/b+/− fish (b(X)) compared to the UAS:GFP control fish (b(IX)). Scale bars: (b(III,IV)): 10 µm; (b(V–VIII)): 100 µm; (b(IX,X)): 1 cm.
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