HECTD3 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to disrupt the HECTD3 gene in human embryonic kidney HEK293T cells. This heterogeneous knockout pool provides a robust loss-of-function model for investigating HECTD3-dependent cellular processes without the need for single-cell cloning, ensuring comprehensive representation of gene-disrupted alleles. The product is optimized for functional studies requiring sustained HECTD3 depletion, enabling dissection of its roles in ubiquitin-mediated signaling, DNA damage response, and NF-??B pathway regulation.
The host cell line, HEK293T, is a HEK293 derivative stably expressing the SV40 large T-antigen. This modification enhances episomal plasmid replication, resulting in high-level protein expression and efficient lentiviral production, making it a premier system for recombinant protein production and functional genomics. HEK293T cells exhibit adherent epithelial morphology, rapid proliferation, and exceptional transfection efficiency, providing an ideal platform for generating gene-edited knockout models to study complex signaling networks in a controlled cellular context.
HECTD3 encodes a HECT-domain E3 ubiquitin ligase that catalyzes K63-linked polyubiquitination, targeting substrates for proteasomal degradation. Mechanistically, it promotes NF-??B activation by ubiquitinating and degrading TRAF3, a negative regulator of NF-??B signaling, and impairs DNA double-strand break repair by ubiquitinating the acetyltransferase Tip60 (KAT5), attenuating its function. HECTD3 activity depends on interactions with UBE2 family E2 enzymes, and its substrates interface with the ATM kinase pathway. Thus, HECTD3 integrates upstream signals from inflammatory cytokines and DNA damage to regulate downstream effectors such as NF-??B p65 and ATM, positioning it at a critical nexus of inflammation, DNA repair, and genomic stability.
In the HEK293T background, polyclonal HECTD3 knockout leads to TRAF3 stabilization, which attenuates NF-??B activation in response to TNF?? or genotoxic agents, while preserving Tip60 activity and enhancing DNA repair capacity. This model allows detailed examination of how HECTD3 coordinates the cellular response to stress and inflammation. The high transfectability of HEK293T cells facilitates biochemical assays such as co-immunoprecipitation and ubiquitination assays, enabling straightforward mapping of HECTD3 substrate interactions and regulatory mechanisms without confounding clonal variation.
Researchers can employ this knockout cell population in diverse applications: NF-??B luciferase reporter assays or RT-qPCR of target genes following cytokine stimulation quantify pathway activity; comet assays and immunofluorescence for ??-H2AX and phospho-ATM assess DNA repair efficiency; western blotting and co-immunoprecipitation validate changes in TRAF3, Tip60, and NF-??B components; flow cytometry monitors cell cycle perturbations or apoptosis. The model is valuable for studying ubiquitin-proteasome system crosstalk with NF-??B signaling and DNA damage responses, and for drug target validation in cancer, neurodegeneration, and immune disorders. For more information on this product, please contact Ascent Research.
