Design of iPSC-based cell model to study the functions of the UBE2A gene

BACKGROUND: The UBE2A protein belongs to the E2 family of ubiquitin-binding enzymes involved in the ubiquitination of substrate proteins. Mutations in the UBE2A gene lead to congenital X-linked mental retardation syndrome type Nascimento. It is still unknown how UBE2A participates in the development of the central nervous system. AIM: We aimed to establish a cell model based on induced pluripotent stem cells (iPSCs) to study the molecular and cellular functions of the UBE2A gene in neurogenesis. METHODS: Using genomic CRISPR-Cas9 editing and lentiviral transduction, we designed a cell model based on IPSCs from two healthy donors. This cell model includes isogenic iPSCs with knockout and inducible hyperexpression of the UBE2A gene. In addition, we obtained iPSCs by reprogramming peripheral blood mononuclear cells of a patient diagnosed with X-linked mental retardation of Nascimento type, which has a deletion spanning the whole UBE2A gene locus. RESULTS: The obtained iPSCs demonstrate ESC-like morphology. They express pluripotent cell markers OCT4, SOX2, SSEA-4, and TRA1-81 and have normal karyotypes. We found that IPSCs with UBE2A gene knockout or hyperexpression had significantly increased nuclei size compared to isogenic control. CONCLUSION: We established the iPSC-based cell model, which can be used for fundamental studies of the UBE2A gene functions in neurogenesis.