TRIM25 Knockout Cell Line (A549)

The choice depends on whether you are studying TRIM25's role in RIG-I-mediated antiviral signaling or its broader functions in ubiquitin ligase biology. The Knockout line is the standard tool for asking whether TRIM25 is required for K63-linked ubiquitination of RIG-I and downstream type I IFN induction — particularly relevant in A-549, which is widely used in respiratory viral infection studies. Overexpression is useful for testing whether elevated TRIM25 enhances antiviral signaling or for studying TRIM25's RNA-binding functions independent of its E3 ligase activity. For antiviral immunity research, the EDITGENE Knockout line in A-549 is highly informative — A-549 supports replication of many respiratory viruses (influenza, SARS-CoV-2, RSV), enabling direct testing of TRIM25's antiviral functions in a disease-relevant context. Rescue with wild-type, RING-dead, or RNA-binding-deficient TRIM25 is the standard approach for dissecting its dual ligase and RNA-binding activities.

Primary applications: • RIG-I ubiquitination assays: Western blot analysis of K63-linked ubiquitination on RIG-I following viral RNA stimulation or poly(I:C) treatment. • Type I IFN induction: IFN-β reporter assays and ISG expression analysis following viral infection (influenza, SARS-CoV-2, Sendai virus) or viral mimics. • Antiviral activity: viral replication assays in A-549 to assess functional consequences of disrupted TRIM25-mediated RIG-I activation. • Substrate identification: TRIM25 ubiquitination targets beyond RIG-I have been reported; ubiquitin proteomics in the knockout can expand the substrate catalog. EDITGENE recommends this model for researchers investigating innate antiviral immunity, respiratory virus infection biology, and RIG-I-MAVS pathway regulation.

Yes. TRIM25 rescue experiments require attention to its dual E3 ligase and RNA-binding activities: • Construct design: use a codon-modified TRIM25 sequence with a small C-terminal tag (FLAG, HA). TRIM25 contains an N-terminal RING domain that should be preserved with minimal N-terminal modification. • RING-dead rescue: the C20A/H22A double mutation in the RING domain abolishes E3 ligase activity and is the standard control for separating ligase from RNA-binding functions. • RNA-binding-deficient rescue: specific PRY/SPRY domain mutations affect TRIM25's RNA binding without disrupting E3 activity — these constructs are critical for dissecting RNA-binding-specific functions. • Functional readout: rescue should restore RIG-I ubiquitination and IFN induction following viral RNA stimulation in A-549. A-549 transduces efficiently with lentivirus and supports stable rescue line generation; the NSCLC background is particularly suited to studying TRIM25 in respiratory virus infection contexts.