Akademik Veri Yönetim Sistemi
8th International Congress of the Molecular Biology Association of Turkey, İstanbul, Türkiye, 9 - 12 Haziran 2022
Background/aim: Due to the increased rate of consanguineous marriages in Turkey and our geography, the incidence of rare genetic disease is higher. A recently identified neurodevelopmental genetic disorder is caused by a point mutation in the splice site consensus sequence of the gene coding for synbindin, a protein involved in vesicular trafficking and autophagy. The mutation results in a splicing defect of the pre-mRNA and substantially reduces the intact, functional protein’s expression level. Several Turkish subjects carrying the mutation homozygous were reported to be affected and they display clinical findings including microcephaly, cerebral atrophy, psychomotor delay, developmental regression and early-onset epilepsy. We have decided to create a mouse model for this disease in order to understand its aetiology and elucidate its molecular mechanism.
Materials and methods: We used CRISPR/Cas9 genome editing technology to edit mouse embryos and introduce a targeted DNA modification that mimics the pathogenic human sequence variant. Cas9 proteins in complex with sgRNA molecules (RNP) were transferred to zygote stage C57BL/6 mouse embryos in the presence of an asymmetric repair template (ssODN) carrying the intended mutation. Edited embryos were transferred to recipient host female mice and new-born mice were genotyped by PCR and restriction analysis.
Results: We have created several genetically modified founder mice that carry the disease mutation (knockin), as well as mice with indel mutations that potentially result in a frameshift and abrogate the protein function (knockout). Heterozygous knockin mice were obtained by backcrossing to C57BL/6 mice. Heterozygous interbreeding is in progress and homozygous pups are expected to be born.
Conclusion: In depth analysis of the homozygous knockin and/or knockout mice will be carried out in order to phenotypically characterize the mechanism of this novel disease and will give insight into the protein’s physiological roles in mammals.