Description

The TLR4 Knockout HEK293T Cell Line is a CRISPR/Cas9-edited knockout cell line featuring targeted disruption of the TLR4 gene, providing a definitive loss-of-function model for dissecting Toll-like receptor 4-mediated innate immune signaling in a human epithelial context. This engineered knockout eliminates functional TLR4 protein expression, enabling researchers to study LPS-induced pathways without interference from endogenous receptor activity and facilitating clean interpretation of downstream molecular events.

HEK293T cells are an immortalized human embryonic kidney cell line that stably expresses the SV40 large T antigen, which promotes high-level transient transfection and robust protein expression. These adherent epithelial cells are widely employed in cell biology, signal transduction, and drug discovery due to their reliable culture characteristics and amenability to a broad range of molecular tools. The HEK293T background thus offers a versatile and well-characterized platform for introducing targeted gene disruptions and assessing signaling consequences.

TLR4 functions as a pattern recognition receptor that specifically senses bacterial lipopolysaccharide (LPS) when complexed with co-receptors MD-2 and CD14. Upon LPS engagement, TLR4 dimerizes and recruits the adaptor proteins MyD88 and TRIF via TIRAP and TRAM, respectively. MyD88-dependent signaling activates IRAK family kinases and TRAF6, leading to NF-??B and AP-1 transcriptional activity, while TRIF-dependent signaling phosphorylates IRF3 to drive type I interferon production. Consequently, TLR4 activation promotes the expression of pro-inflammatory cytokines including TNF??, IL-6, and IL-1??, along with interferon-responsive targets such as IFN?? and ICAM1.

The HEK293T epithelial background, with its high transfection efficiency and capacity for exogenous protein production, makes this TLR4 knockout cell line especially valuable for reconstitution experiments and structure-function analyses. Re-introduction of wild-type or mutant TLR4 variants allows precise mapping of receptor domains and signaling checkpoints. Additionally, the absence of TLR4 provides a clean system to evaluate contributions of other innate immune sensors that may respond to DAMPs such as HMGB1 or heat shock proteins, thereby clarifying receptor-specific roles in inflammatory pathways.

This knockout cell line supports diverse experimental workflows, including LPS stimulation assays coupled with NF-??B luciferase reporter measurements, quantitative RT-qPCR analysis of IL6 and TNF?? mRNA induction, western blotting for TLR4 and phospho-NF-??B, ELISA-based quantification of secreted cytokines, and immunofluorescence to visualize NF-??B p65 nuclear translocation. It serves as an essential negative control for TLR4-dependent effects and is well-suited for high-throughput screening of TLR4 inhibitors. For further technical information or to request a quotation, please contact Ascent Research.