The CD274 Knockout HEK293 Polyclonal Cells provide a CRISPR/Cas9-edited polyclonal knockout population in which the CD274 gene, encoding the programmed death-ligand 1 (PD-L1) protein, has been functionally disrupted. This loss-of-function model enables researchers to interrogate PD-L1 biology in a well-characterized human embryonic kidney epithelial cell background, free from endogenous PD-L1 expression. The polyclonal nature of the knockout cells preserves genetic heterogeneity, reducing the risk of clonal artifacts and offering a robust system for studying PD-L1-dependent signaling and immune checkpoint regulation.
Derived from human embryonic kidney cells immortalized by adenovirus 5 DNA transformation, the HEK293 host cell line is a cornerstone of molecular and cellular biology. Its ease of culture, high transfection efficiency, and capacity for protein production make it an ideal platform for genetic manipulation and functional assays. As epithelial cells, HEK293 cells express a repertoire of signaling molecules relevant to PD-L1 regulation, including receptors for interferons, tumor necrosis factor, and epidermal growth factor, allowing mechanistic studies of upstream pathways that control CD274 transcription and surface presentation.
CD274/PD-L1 is a critical immune checkpoint molecule that, upon binding to its receptor PD-1 (PDCD1) on T cells, transmits an inhibitory signal that dampens T cell receptor (TCR) signaling and cytokine production. The PD-L1/PD-1 axis promotes immune tolerance and is frequently exploited by tumors for immune evasion. The signaling network involves recruitment of the tyrosine phosphatase SHP-2 (PTPN11) downstream of PD-1, which dephosphorylates key TCR-associated kinases such as ZAP70 and LCK, ultimately attenuating activation of AKT1 and MTOR pathways. CD274 is transcriptionally regulated by a variety of stimuli, including cytokines like IFNG and TNF, growth factors like EGF, and transcription factors such as STAT1, STAT3, MYC, HIF1A, and IRF1. It also interacts with CD80, providing an additional layer of immune modulation. Representative pathway components thus span CD274, PDCD1, PTPN11, ZAP70, LCK, AKT1, and MTOR.
In the HEK293 background, CD274 knockout disrupts the endogenous PD-L1 expression, allowing for precise reconstruction of PD-L1 variants, analysis of PD-L1-dependent signaling in co-culture with T cell models, and screening of inhibitors that target the PD-L1/PD-1 interaction or PD-L1 expression. The polyclonal cells serve as a versatile tool for both loss-of-function studies and as a parental line for re-expression experiments. Their application is particularly valuable in high-throughput formats where uniform gene disruption across a mixed population is sufficient, such as flow cytometry-based binding assays or functional reporter systems.
Typical research applications include dissecting the molecular mechanisms governing PD-L1 expression in response to upstream regulators like IFNG, investigating PD-L1/PD-1 immune checkpoint signaling in co-culture with PD-1-expressing reporter T cells, and performing drug screening for small molecules or antibodies that modulate PD-L1 levels. Representative assays compatible with this model are western blotting, flow cytometry for surface PD-L1, RT-qPCR for CD274 mRNA quantification, immunofluorescence staining, and luciferase-based NFAT reporter assays to measure T cell activation. For further information, contact Ascent Research.
