The CD274 Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line generated from the murine RAW 264.7 macrophage parental line. It provides a stable loss-of-function model for CD274 (PD-L1), a key immune checkpoint ligand. By disrupting endogenous CD274 expression, this cell line enables investigation of PD-L1-dependent immune regulation without confounding effects of antibody blockade, offering a clean genetic system for functional studies.
RAW 264.7 cells are an extensively characterized mouse macrophage line derived from BALB/c mouse ascites fluid after Abelson leukemia virus transformation. They retain hallmark macrophage functions, including robust phagocytosis, LPS-inducible inflammatory cytokine production, and responsiveness to IFN-?? stimulation. These properties make RAW 264.7 cells a gold-standard model for studying macrophage biology, innate immune signaling, and host?Cpathogen interactions, and they are particularly suitable for examining the role of PD-L1 in macrophage-mediated immune modulation.
CD274 encodes programmed death-ligand 1, which upon binding to its receptor PD-1 (PDCD1) on T cells, triggers recruitment of SHP-2 phosphatase (PTPN11). SHP-2 dephosphorylates proximal TCR signaling molecules, thereby attenuating the PI3K/AKT and RAS/ERK pathways, culminating in diminished T cell proliferation, cytokine output, and cytolytic activity. CD274 expression is transcriptionally upregulated by IFN-?? via the IFNGR?CJAK1?CSTAT1 axis and by NF-??B. Additionally, PD-L1 can interact with B7-1 (CD80), providing cross-regulation of costimulatory signals. Thus, CD274 is a central node in immune checkpoint control.
In RAW 264.7 macrophages, CD274 is dynamically induced by inflammatory stimuli, mimicking the activation-induced upregulation observed in primary macrophages and tumor-associated macrophages. Knocking out CD274 eliminates PD-L1-mediated suppressive capacity, permitting precise dissection of how macrophage PD-L1 contributes to T cell exhaustion, immune tolerance, and tumor immune evasion. This model is highly relevant for studying the macrophage?CT cell interface in cancer, chronic infection, and autoimmunity, where aberrant PD-L1 expression drives immunosuppressive microenvironments.
Typical applications include flow cytometry to confirm PD-L1 ablation, western blotting for downstream effectors like phospho-AKT, and co-culture assays with T cells monitored by ELISA for cytokines such as IL-2 and IFN-??. The line is also suited for PD-1 binding assays, drug screening against PD-L1 inhibitors, and RNA-seq transcriptomic profiling. Collectively, these uses support target validation in immuno-oncology, mechanistic studies of immune checkpoint pathways, and development of macrophage-targeted immunotherapies. For further details, contact Ascent Research.
