UBE3A Knockout Cell Line (HEK293)

The choice depends on whether you are studying UBE3A's role in ubiquitin ligase biology or modeling Angelman syndrome / 15q11-q13 duplication disorders. The Knockout line is the standard tool for both — Angelman syndrome is caused by loss of maternal UBE3A expression. Overexpression is useful for studying 15q duplication syndromes where elevated UBE3A is the disease-driving change. For UBE3A research, the EDITGENE Knockout line in HEK293 provides a tractable mechanistic platform for dissecting substrate ubiquitination and ligase activity, though it is not a neuronal context — UBE3A's most disease-relevant functions are in neurons. Rescue with wild-type, catalytically-dead (C840A), or disease-associated mutant UBE3A is informative for genotype-function correlation.

Primary applications: • Substrate stability assays: cycloheximide chase for reported UBE3A substrates including HHR23A, BAK, p53, and Angelman syndrome-relevant proteins (Arc, Ephexin5, GAT1). • Ubiquitination assays: in vitro and cellular ubiquitination of UBE3A substrates using wild-type and catalytically-dead enzymes. • Disease mutation modeling: rescue with Angelman syndrome-associated UBE3A mutations (T106A, R417X, etc.) to study mutation-specific effects on ligase activity. • Substrate identification: ubiquitin proteomics to expand the UBE3A substrate landscape beyond established targets. EDITGENE recommends this model for researchers investigating UBE3A ubiquitin ligase biology, Angelman syndrome mechanisms, and 15q duplication-related research.

Yes. UBE3A rescue experiments require careful attention to imprinting and dosage considerations: • Construct design: use a codon-modified UBE3A sequence with a C-terminal tag (FLAG, HA). UBE3A's HECT domain at the C-terminus contains the active site cysteine — bulky C-terminal tags should be avoided in favor of small epitope tags. • Catalytically-dead rescue: the C840A mutation in the active site cysteine abolishes ubiquitin ligase activity and is the standard specificity control. • Isoform considerations: UBE3A has three protein isoforms with distinct N-termini and partially different subcellular distributions — rescue with specific isoforms can address isoform-specific functions. • Dosage matters: UBE3A overexpression beyond endogenous levels can produce 15q duplication-like phenotypes — titrate to physiological levels using inducible systems for Angelman syndrome-focused research. HEK293 transduces efficiently with lentivirus and supports stable rescue line generation.