TNFRSF9 Knockout Cell Line (HEK 293)

Note: TNFRSF9 is the receptor (4-1BB/CD137), not the ligand 4-1BBL/TNFSF9. The choice depends on whether you are studying CD137 biology in immune cells or using HEK293 as a heterologous expression platform. Important context: CD137 (4-1BB) is normally expressed on activated T cells, NK cells, and other immune effector cells — HEK293 is not a physiologically relevant CD137-expressing cell type. The EDITGENE Knockout in HEK293 is most useful for biochemical applications: receptor binding studies, anti-CD137 antibody specificity validation, and structural research with heterologously expressed CD137. Overexpression is generally more functionally informative than knockout for CD137 research in HEK293, since heterologous CD137 expression is the standard approach for ligand-binding and antibody characterization studies. The EDITGENE Knockout is useful as a negative control for confirming on-target activity of anti-CD137 immunotherapy agents (urelumab, utomilumab, and newer agonists). Rescue with wild-type CD137 establishes baseline binding characteristics for comparing engineered variants.

Primary applications: • Anti-CD137 antibody specificity: critical genetic control for confirming on-target activity of CD137 agonist antibodies (urelumab, utomilumab) and newer 4-1BB-targeting immunotherapies. • Receptor binding studies: in vitro binding assays for 4-1BBL and engineered CD137-targeting agents using cells producing tagged CD137 variants. • Structural research: heterologous CD137 expression for protein purification and structural biology applications. • Bispecific antibody validation: CD137 is a common target in T-cell-engaging bispecific antibodies; the knockout serves as the negative control arm. EDITGENE recommends this model for researchers in cancer immunotherapy development, particularly for CD137-targeting agents and bispecific antibody validation. Note that functional CD137 costimulation assays require T cells, NK cells, or other immune effector cells, not HEK293.

Yes. CD137 rescue experiments require attention to its surface receptor topology: • Construct design: use a codon-modified TNFRSF9 sequence with a C-terminal tag (HA, FLAG) on the cytoplasmic domain. CD137 is a type I membrane protein — the N-terminal signal peptide and cysteine-rich extracellular domain must be preserved. • Surface expression validation: confirm exogenous CD137 surface localization by flow cytometry with anti-CD137 antibodies before functional assays. • Signaling domain mutant rescue: cytoplasmic TRAF-binding motif mutations dissect ligand-binding from intracellular signaling functions. • Functional readout: rescue should restore antibody and 4-1BBL binding (flow cytometry, SPR/BLI binding assays). HEK293 transduces efficiently with lentivirus and is a standard heterologous expression system for type I membrane receptor characterization.