TGFBI Geninde Genom Düzenleme Tekniği ile Oluşturulan Varyasyonların Zebra Balığı Korneasındaki Fenotipik Etkilerinin İncelenmesi
Özet
YAYLACIOĞLU TUNCAY F. Evaluation of Phenotypic Effects of Genome Editing-mediated TGFBI Variation on Zebrafish Cornea. Hacettepe University, Graduate School of Health Sciences, Medical Biology PhD Thesis, Ankara, 2019. TGFBI-related corneal dystrophies are hereditary disorders characterized by accumulation of the abnormal protein product of TGFBI gene in the hyaline or amyloid form due to missense mutations of this gene. This gene exists in the zebrafish genome without duplication and its protein product has 65% homology to its human ortholoque. Protein product of tgfbi (TGFBIp) was shown in the cornea of zebrafish. Mutations of this gene that effect the arginine residue on the 124th position of TGFBIp were reported as one of the hot spots for this group of corneal dystrophies. This argininine residue was also conserved in zebrafish. According to these data; in this study we aimed to make an in frame change in the region that codes the 124th amino acid residue of tgfbi in the zebrafish genome by using TALEN or CRISPR/Cas9. Finally, we achieved indel variation at the target sequence that resulted in p. Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair. This variation could mimic the disease process in the zebrafish cornea. The 3 months old zebrafish eyes were examined histopathologically under the light microscope but no amiloid or hyaline deposits could be detected with hematoxylen-eosin, masson-trichrome and congo red staining. Thus, this study is the first in literature that succeeded to make an in frame variation effecting the hot spot arginine residue in tgfbi protein and further studies could detect phenotypical changes in older zebrafish that could mimic the TGFBI-related corneal dystrophies and help to reveal the disease pathogenesis.
