The Rfx5 Knockout CT26 Cell Line is a CRISPR/Cas9-edited knockout cell line featuring a targeted disruption of the Rfx5 gene in the murine CT26 colon carcinoma background. Rfx5 encodes a critical subunit of the regulatory factor X (RFX) complex, a master transcriptional regulator of major histocompatibility complex class II (MHC class II) gene expression and adaptive immunity. This cell line serves as a genetically defined model to dissect the role of Rfx5-dependent antigen presentation in tumor biology and immune surveillance.
The CT26 cell line was derived from a chemically induced colon carcinoma in a BALB/c mouse and is widely used as a syngeneic tumor model in cancer immunotherapy and tumor immunology. These immunogenic cells upregulate MHC class II upon interferon-gamma (IFNG) stimulation via the JAK-STAT-IRF1-CIITA pathway, enabling CD4+ T cell activation. Consequently, CT26 provides an optimal background for studying the interplay between MHC class II-dependent antigen presentation and anti-tumor immunity.
Rfx5 is a DNA-binding subunit of the RFX complex, together with RFXAP and RFXANK, which cooperates with CIITA to drive MHC class II transcription. IFNG receptor engagement activates JAK1/JAK2, leading to STAT1 phosphorylation and induction of IRF1 and CIITA. CIITA then recruits the RFX complex and NF-Y to MHC class II promoters and the CD74 gene. Disruption of Rfx5 abrogates this enhanceosome, eliminating expression of MHC class II alleles (e.g., H2-Aa, H2-Eb) and CD74, thereby impairing CD4+ T cell-mediated immune responses.
In CT26 cells, Rfx5 knockout abolishes IFNG-induced MHC class II surface expression, preventing tumor cell recognition by CD4+ T cells. This mimics bare lymphocyte syndrome type II and provides a platform to investigate immune evasion through MHC class II downregulation. The model enables dissection of CD4+ T cell-specific contributions to tumor rejection, checkpoint blockade efficacy, and tumor microenvironment, while facilitating study of alternative antigen cross-presentation pathways.
This knockout cell line is validated for flow cytometry, western blotting for RFX5, phospho-STAT1, and CIITA, and RT-qPCR for MHC class II and CD74 transcripts. Co-culture with CD4+ T cells and cytokine profiling (e.g., IL-2, IFNG) assess functional antigen presentation. In vivo syngeneic tumor implantation in BALB/c mice enables monitoring of tumor growth, immune infiltration, and immunotherapy responses. The Rfx5 Knockout CT26 Cell Line serves as a versatile tool for preclinical immuno-oncology research. For further information, please contact Ascent Research.
