The ISG15 Knockout HT29 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population targeting the ISG15 gene in the human HT29 colorectal adenocarcinoma epithelial cell line. This loss-of-function model is generated through CRISPR/Cas9-mediated gene disruption of ISG15, resulting in a heterogeneous pool of cells with ablated ISG15 expression. The polyclonal format provides a robust and representative population for studying ISG15-dependent functions without the clonal selection biases inherent in single-cell-derived lines.
The HT29 cell line serves as a well-established model of colorectal adenocarcinoma, originally isolated from a 44-year-old female patient. These cells exhibit adherent epithelial morphology, retain microsatellite stability, and carry a mutation in the tumor suppressor TP53. HT29 cells are extensively used to study intestinal epithelial biology, including barrier function, mucus production, and tumor progression. Their epithelial nature and responsiveness to cytokines make them an ideal host for interrogating interferon and inflammatory signaling pathways.
ISG15 encodes a ubiquitin-like protein that is rapidly induced by type I interferons (IFN-??/??) and conjugated to target proteins through an ISGylation cascade involving the E1 enzyme UBE1L, E2 enzyme UBCH8, and E3 ligases such as HERC5. ISG15 conjugation modulates the stability and activity of key signaling molecules including IRF3, STAT1, JAK1, and filamin B, thereby regulating antiviral innate immune responses and interferon signaling. ISG15 is also secreted as a cytokine, contributing to immunomodulation. Upstream activation is mediated by transcription factors IRF3, IRF7, STAT1, STAT2, and NF-??B, which bind to interferon-stimulated response elements (ISRE) in the ISG15 promoter. The deISGylating enzyme USP18 provides a critical counterbalance by removing ISG15 from substrates. ISG15 thus integrates signals from JAK-STAT and NF-??B pathways to coordinate cellular antiviral defenses and inflammatory responses.
In the HT29 colorectal cancer context, ISG15 knockout disrupts interferon-induced ISGylation, impairing antiviral signaling and potentially altering tumor cell proliferation, migration, and immune evasion. Given that colorectal cancer cells often encounter inflammatory microenvironments and viral triggers, this knockout model enables dissection of ISG15’s dual roles in epithelial tumor biology and innate immunity. The loss of ISG15 may affect growth characteristics, response to interferon stimulation, and interactions with immune effectors, offering insights into cancer cell adaptability and therapeutic vulnerabilities.
Researchers can employ the ISG15 Knockout HT29 Polyclonal Cells to investigate the role of ISGylation in interferon response, antiviral pathways, and tumor cell signaling. Typical applications include western blotting for ISG15 and ISGylation substrates, RT-qPCR for ISG15 and interferon-stimulated genes, interferon stimulation followed by phospho-STAT1 analysis, co-immunoprecipitation of ISG15 conjugates, and functional assays such as cell proliferation (MTS/MTT), migration/invasion (transwell), and flow cytometry for surface markers. Additionally, these cells are suitable for RNA-seq profiling of interferon-treated populations and antiviral response assays. This polyclonal knockout population provides a versatile tool for high-content screening and mechanistic studies. For further technical details, please contact Ascent Research.
