|
Fig. 1
Somatic inactivation of cdkn2a/b gene by the injection of TALENs mRNA leads to MPNSTs in F0 founder zebrafish. (A) The targeted regions of two different TALENs (cdkn2a/b TALENs-1 and cdkn2a/b TALENs-2) are located in exon 1 of cdkn2a/b gene. The binding sequences of TALENs for cdkn2a/b gene are also shown. (B) By the injection of cdkn2a/b TALENs-1 mRNA, 7 different germline mutated alleles of cdkn2a/b gene were recovered at F1 generation. And 9 different alleles were also recovered at F1 generation after injection of cdkn2a/b TALENs-2 mRNA. (C) The large mass of tumor was observed in adjacent to eyes of the cdkn2a/b TALENs mRNA injected tp53 mutant zebrafish at 5.5 month post fertilization. (D–F) Histological image of cross sections of tumor tissue was visualized with Hematoxylin-Eosin staining. (D) High magnification image of tumor tissue shows the herringbone pattern that is a typical feature of malignant peripheral nerve sheath tumor. (F) There is no evident abnormality in the neuroepithelial layer of retina adjacent to tumor tissue. (G) Kaplan-Meier survival representation of wild type (AB), tp53 mutant, and cdkn2a/b TALENs mRNA injected AB or tp53 mutant zebrafish. The cdkn2a/b TALENs mRNA injected tp53 mutant zebrafish had only started to exhibit death with tumor bearing as early as about 5 months. Other adult zebrafish such as tp53 mutant and cdkn2a/b TALENs mRNA injected AB zebrafish did not show early death with tumor bearing. All data was acquired from two independent experiments. Scale bars: 2 mm (E) and 100 μm (D and F).