BACKGROUND:Spinocerebel ar ataxia type 3 (SCA3) is a typical genetic neurodegenerative disease. To establish patient-specific disease models of genetic background contributes to studying the pathogenesis and exploring therapeutic manners.
OBJECTIVE:To observe the effectiveness of neural differentiation of induced pluripotent stem cel lines induced by SCA3 and the stability of CAG copy number.
METHODS:Skin tissue of SCA3 patient was obtained clinical y, and specific skin flbroblasts were isolated and cultured. Reprogramming fibroblasts could obtain induced pluripotent stem cel s. Patient-specific induced pluripotent stem cel s, normal person induced pluripotent stem cel s (NHF) and embryonic stem cel s (ES-10) were induced to differentiate. Flow cytometry was used to compare the efficiency of differentiation. Western blot assay was utilized to detect ataxin-3 protein expression in neurons. Polymerase chain reaction was applied to measure the CAG repeat number of SCA3/ATXN3 gene.
RESULTS AND CONCLUSION:Induced pluripotent stem cel s that had identical genetic background to fibroblasts were successful y obtained, and had similar morphology and multi-directional differentiation potential to human embryonic stem cel s. Each cel line could differentiate into neural stem cel s. The CAG number did not apparently alter before and after reprogramming as wel as induction of neuronal differentiation. The effectiveness of the differentiation of induced pluripotent stem cel s derived from SCA3 into neural stem cel s was lower than that of normal person-derived induced pluripotent stem cel s (NHF) and embryonic stem cel s (ES-10). These findings demonstrate that reprogramming can successful y establish human induced pluripotent stem cel s, and induced the differentiation of above cel s into neural stem cel s. In the whole process, CAG number did not obviously alter, which was consistent with body cel s of patients.

The CRISPR-Cas9 system is a new targeted nuclease for genome editing, which can directly introduce modifications at the targeted genomic locus. The system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 in the genome. Upon targeting, Cas9 can generate DNA double-strand breaks (DSBs). As such DSBs are repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR), therefore facilitates introduction of random or specific mutations, repair of endogenous mutations, or insertion of DNA elements. The system has been successfully used to generate gene targeted cell lines including those of human, animals and plants. This article reviews recent advances made in this rapidly evolving technique for the generation of animal models for human diseases.
Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; genetics ; Disease Models, Animal ; Humans ; RNA Editing ; genetics