The HECTD3 Knockout HeLa Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population engineered to disrupt the HECTD3 gene in HeLa cells. This heterogeneous pool offers a loss-of-function model for exploring HECTD3-dependent signaling, avoiding the biases inherent to clonal isolates. CRISPR/Cas9-mediated targeting enables robust assessment of gene function in a cancer-relevant cellular background.
The HeLa cell line, an immortalized human cervical adenocarcinoma line positive for HPV18, is extensively employed in cancer research and innate immunity studies. These epithelial cells retain functional STING-dependent interferon pathways, though HPV E6 and E7 oncoproteins partially attenuate antiviral responses, making them a pertinent model for investigating immune evasion mechanisms. The genetic tractability of HeLa cells facilitates the generation of knockout models for dissecting ubiquitin ligase biology.
HECTD3 encodes a HECT-domain E3 ubiquitin ligase that negatively regulates innate antiviral immunity. Mechanistically, HECTD3 catalyzes K27-linked polyubiquitination of STING (TMEM173) at lysine 288 and TRAF3, targeting both adaptors for proteasomal degradation. This dual ubiquitination suppresses the activation of the kinases TBK1 and IKK??, thereby inhibiting phosphorylation of the transcription factors IRF3 and NF-??B. Consequently, the induction of type I interferons like IFN?? and interferon-stimulated genes is reduced. HECTD3 expression itself is upregulated by inflammatory cytokines IL-1 and TNF-??, as well as by NF-??B, establishing a negative feedback loop that constrains immune signaling. This central role places HECTD3 at a critical node in STING- and TRAF3-mediated pathways.
In the HeLa context, loss of HECTD3 is expected to enhance STING and TRAF3 protein stability, potentiating innate immune responses. This model is particularly valuable for studying how cervical cancer cells modulate interferon and NF-??B signaling, pathways often subverted by HPV to promote viral persistence and tumor progression. Furthermore, HECTD3 overexpression has been associated with gastric and hepatocellular carcinomas, implicating it in tumorigenesis; therefore, these knockout cells can be used to examine its role in cancer cell proliferation and immune surveillance. The polyclonal population captures a broad range of knockout phenotypes, facilitating robust phenotypic comparisons.
These polyclonal knockout cells are suitable for a comprehensive array of experimental applications. Standard assays include western blotting to detect HECTD3, STING, TRAF3, and phosphorylated IRF3; RT-qPCR for interferon-stimulated genes such as IFIT1 and ISG15; and IFN?? luciferase reporter assays to quantify pathway activation. Co-immunoprecipitation experiments enable direct assessment of STING and TRAF3 ubiquitination changes. Viral infection assays using HSV-1 or VSV serve as physiological readouts of antiviral competence, while cell viability and proliferation assays evaluate the impact of HECTD3 loss on cancer cell growth. For technical inquiries or customization requests, please contact Ascent Research.
