The ISG15 Knockout DLD-1 Polyclonal Cells represent a CRISPR/Cas9-mediated gene-disrupted polyclonal population in the DLD-1 human colorectal adenocarcinoma cell line, providing a stable loss-of-function model for the ISG15 locus. This polyclonal knockout cell product is generated by transient delivery of Cas9 and a target-specific guide RNA, resulting in a heterogeneous pool of cells harboring targeted gene disruption. The polyclonal format retains inherent biological variability while ensuring functional ablation of ISG15 expression, making it suitable for population-level studies of interferon signaling, antiviral responses, and colorectal cancer biology without the bias of single-cell cloning. The cells are supplied as a ready-to-use culture, facilitating direct integration into downstream phenotypic and mechanistic assays.
DLD-1 is a well-characterized human colorectal adenocarcinoma epithelial cell line established from a colorectal carcinoma with microsatellite instability and mutations in APC and TP53. It exhibits an epithelial morphology and is widely employed as a model for colorectal cancer pathogenesis, drug screening, and signal transduction investigations. The cell line maintains key features of intestinal epithelium and recapitulates oncogenic signaling aberrations commonly observed in colorectal tumors, including altered Wnt/??-catenin, TGF-??, and p53 pathways. Its robust growth characteristics and genetic tractability make it a preferred host for gene-editing approaches aimed at dissecting molecular mechanisms in colon cancer.
ISG15 encodes a 15-kDa ubiquitin-like protein that is strongly induced by type I interferon (IFN-??/??) and, to a lesser extent, interferon-??. Upstream regulators include transcription factors IRF3, IRF7, and NF-??B, which are activated downstream of pattern recognition receptors such as RIG-I and MDA5. ISG15 becomes covalently conjugated to target proteins through an ISGylation cascade involving the E1 enzyme UBE1L, E2 enzyme UbcH8, and the major E3 ligase HERC5. This post-translational modification modulates the stability, localization, or function of numerous substrates, including IRF3, STAT1, p53, RIG-I, and TRIM25, thereby shaping type I interferon signaling, antiviral effector mechanisms, and inflammatory responses. DeISGylation is mediated by the protease USP18, creating a dynamic regulatory system. ISG15 also has unconjugated functions, acting as a secreted cytokine that stimulates NK cell proliferation and IFN-?? production.
Within the DLD-1 colorectal adenocarcinoma context, ISG15 knockout provides a powerful tool to investigate tumor cell-intrinsic roles of the ISGylation pathway. ISG15 expression has been implicated in modulating proliferation and apoptosis in cancer cells, and its deletion allows researchers to assess how ISGylation status affects colorectal tumorigenesis, chemosensitivity, and immunogenicity. Given that DLD-1 cells harbor a mutant p53 background, the ISG15?Cp53 axis is of particular interest, as ISG15 can influence p53 transcriptional activity and stability. Additionally, the knockout model permits dissection of interferon-driven tumor-suppressive or tumor-promoting signals, advancing the understanding of immune checkpoint regulation in the colorectal cancer microenvironment.
This knockout cell product supports a wide range of experimental applications, including western blotting for ISG15 and ISGylation, RT-qPCR profiling of interferon-stimulated genes such as MxA and OAS1, RNA-seq transcriptome analysis, immunofluorescence for protein localization, co-immunoprecipitation to capture ISG15 conjugates, and flow cytometry for apoptosis and cell cycle distribution. Viral infection assays enable investigation of ISG15-dependent antiviral restriction, while phospho-signaling analysis (e.g., STAT1 phosphorylation) delineates interferon pathway readouts. Colony formation assays facilitate evaluation of clonogenic survival. These applications are directly relevant to antiviral innate immunity research, interferon signaling pathway dissection, colorectal cancer pathogenesis studies, identification of novel ISG15 substrates, and drug target validation. For further technical details, please contact Ascent Research.
