IFIH1 Knockout Jurkat Polyclonal Cells provide a CRISPR/Cas9-edited polyclonal knockout cell population in the Jurkat T lymphocyte background, with targeted disruption of the IFIH1 gene encoding the RIG-I-like receptor MDA5. This product comprises a heterogeneous pool of Jurkat cells carrying mutations at the IFIH1 locus, generated via non-clonal CRISPR/Cas9-mediated gene disruption, allowing loss-of-function analyses of MDA5-dependent innate immune signaling. The polyclonal format preserves cellular diversity while ablating MDA5 function, making it suitable for investigating antiviral pathways in a human T cell context.
The Jurkat host cell line is an immortalized human T lymphocyte line originally derived from the peripheral blood of a 14-year-old male with T cell acute lymphoblastic leukemia. Jurkat cells grow in suspension and serve as a widely established model for studying T cell receptor signaling, immune response mechanisms, and lymphocyte biology. They express key components of the innate antiviral signaling machinery, including the RIG-I-like receptor pathway, rendering them a relevant system for dissecting MDA5-mediated responses.
MDA5, encoded by IFIH1, functions as a cytosolic sensor of long double-stranded RNA and is activated by viral dsRNA or synthetic mimics such as poly(I:C). Upon ligand recognition, MDA5 interacts with the adaptor protein MAVS through CARD domain interactions, triggering the assembly of a signaling complex that recruits the kinases TBK1 and IKK??. These kinases phosphorylate the transcription factors IRF3 and NF-??B, which translocate to the nucleus and drive the expression of type I interferons, including IFN-??, and downstream interferon-stimulated genes (ISGs). MDA5 activity is regulated by interacting factors such as LGP2, TRIM25, 14-3-3 proteins, and PACT, and its expression is induced by type I interferons via STAT1/STAT2/IRF9 signaling, forming a positive feedback loop. In Jurkat cells, this pathway connects viral RNA detection to T cell-intrinsic antiviral effector programs.
Loss of MDA5 in Jurkat polyclonal cells abrogates MAVS-dependent signal transduction, leading to a failure in IRF3 and NF-??B activation upon viral challenge or dsRNA stimulation. Consequently, IFIH1 knockout Jurkat cells exhibit severely impaired production of type I interferons and pro-inflammatory cytokines, recapitulating the signaling defects observed in MDA5-deficient primary immune cells. This knockout model enables the specific interrogation of MDA5??s role in T lymphocyte innate immunity, distinguishing it from the related RIG-I pathway. The polyclonal nature of the population mitigates artifacts from clonal selection while providing a robust loss-of-function phenotype for mechanistic studies.
Typical applications include dissection of antiviral signaling networks in T cells, validation of MDA5-specific functions downstream of viral infection or dsRNA treatment, and investigation of interferon induction mechanisms using assays such as Western blotting for phosphorylated IRF3 and NF-??B, RT-qPCR for IFN-?? and ISGs, ELISA for cytokine secretion, and reporter assays with IFN-?? promoter luciferase constructs. These cells are also amenable to flow cytometric analysis of activation markers, RNA-seq transcriptome profiling, and phospho-signaling studies. Moreover, they can be used in drug screening to identify modulators of the RLR pathway and in autoimmune disease models relevant to Aicardi-Gouti??res syndrome, Singleton-Merten syndrome, and systemic lupus erythematosus, where MDA5 gain-of-function mutations drive pathology. For further technical information or to inquire about custom applications, please contact Ascent Research.
