ISG15 Knockout HCT 116 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population in which the ISG15 gene has been disrupted to create a loss-of-function model. This product provides a heterogeneous pool of HCT 116 cells carrying diverse ISG15 inactivation events, enabling robust study of ISG15-dependent phenotypes without clonal artefacts. The polyclonal format ensures representation of the full spectrum of editing outcomes typical of CRISPR/Cas9-mediated gene disruption, making it suitable for experiments requiring population-level analysis of ISG15 function. As a research tool, it is designed for investigating the multifaceted roles of ISG15 in interferon signaling, ISGylation, antiviral innate immunity, and cancer biology.
HCT 116 is a human colorectal carcinoma cell line with epithelial morphology and microsatellite instability (MSI), widely employed as a model for colorectal cancer research and drug discovery. This cell line exhibits intact interferon signaling pathways and is responsive to both type I and type II interferons, facilitating detailed dissection of interferon-stimulated gene networks. The MSI status and well-characterized genetic background, including mutations in KRAS and MLH1, make HCT 116 particularly valuable for studying tumor immunology, DNA damage responses, and the interplay between oncogenic signaling and innate immunity. The knockout of ISG15 in this context enables analysis of how an interferon-induced ubiquitin-like modifier influences cancer cell behavior and immune evasion.
ISG15 is an interferon-induced ubiquitin-like protein that becomes conjugated to target proteins through an enzymatic cascade involving UBE1L (E1), UbcH8 (E2), and HERC5 or HERC6 (E3 ligases), a process known as ISGylation. This reversible modification is countered by the deconjugating enzyme USP18, establishing a dynamic regulatory system. ISG15 is transcriptionally induced by type I interferons (IFN-??/??) and IFN-?? via the JAK-STAT pathway, with key transcription factors including IRF3, IRF7, STAT1, STAT2, and IRF9. Downstream, ISG15 conjugates to a broad set of substrates, including JAK1, STAT1, RIG-I, MDA5, and PKR, modulating their stability, activity, and subcellular localization. Through these interactions, ISG15 governs antiviral innate immune responses, regulates JAK-STAT signaling, and influences protein stability within the ubiquitin-proteasome system, while also participating in immune modulation via extracellular ISG15 binding to LFA-1 integrin.
In the HCT 116 background, ISG15 knockout provides a powerful system to dissect tumor cell-intrinsic roles of ISGylation in colorectal carcinoma. The model allows investigation of how loss of ISG15 impacts interferon-driven gene expression programs, STAT phosphorylation kinetics, and the cellular response to oncolytic viruses or interferon-based therapies. Given the MSI phenotype of HCT 116, researchers can explore ISG15??s contributions to DNA repair fidelity, mutation burden, and sensitivity to immune checkpoint inhibition, as well as its role in regulating tumor-infiltrating lymphocyte activity through extracellular ISG15. This knockout model thus bridges innate antiviral immunity and cancer cell biology, enabling mechanistic studies of ISG15 in tumor progression and immunomodulation.
Typical applications include Western blotting for ISG15 and its conjugates to assess ISGylation status, RT-qPCR for ISG15 mRNA induction following interferon stimulation, and phospho-STAT flow cytometry to quantify JAK-STAT signaling activity. Co-immunoprecipitation experiments can identify novel ISGylated proteins, while RNA-seq enables transcriptomic profiling of interferon-responsive gene networks. Cell viability and interferon stimulation assays are valuable for evaluating drug sensitivity and antiviral states. This polyclonal knockout cell population is particularly suited for large-scale functional screens, pathway epistasis experiments, and studies of tumor immunology where population-level responses are critical. For more information and technical support, please contact Ascent Research.
