SIRT5 Knockout Cell Line (HEK293)

The choice depends on whether you are studying SIRT5's role as a unique mitochondrial desuccinylase/demalonylase/deglutarylase or its emerging functions in metabolic regulation and cancer. The Knockout line is the standard tool for asking whether SIRT5 is required for removing these acidic PTMs from mitochondrial substrates — SIRT5 is the only mammalian sirtuin with substantial desuccinylation/demalonylation activity, distinguishing it functionally from the other six sirtuins. Overexpression is useful for testing SIRT5 in metabolic stress contexts or for assessing its role in cancer biology. For sirtuin research, the EDITGENE SIRT5 Knockout in HEK293 is the standard mechanistic platform — HEK293 supports the biochemical and proteomic analyses needed for SIRT5 substrate characterization. Rescue with wild-type or catalytically-dead (H158Y) SIRT5 is the standard specificity control. The knockout serves as a critical genetic specificity tool for SIRT5-selective compounds in development.

Primary applications: • Mitochondrial PTM profiling: lysine succinylation, malonylation, and glutarylation analysis by mass spectrometry — these PTMs accumulate in SIRT5-null mitochondria. • Metabolic enzyme regulation: SIRT5 substrates (CPS1, SOD1, GAPDH, PKM2, others) activity and acidic acyl-modification status analysis. • In vitro desuccinylation: SIRT5 enzymatic activity assays using succinyl-peptide substrates with NAD⁺ supplementation. • Cancer biology: proliferation, metabolic stress sensitivity assays given SIRT5's reported roles in tumor cell metabolism. EDITGENE recommends this model for researchers investigating SIRT5 biology, mitochondrial acidic PTM regulation, and metabolic enzyme post-translational modifications.

Yes. SIRT5 rescue experiments require attention to mitochondrial targeting and catalytic activity: • Construct design: use a codon-modified SIRT5 sequence with a small C-terminal tag (FLAG, HA). SIRT5 contains an N-terminal mitochondrial targeting sequence — N-terminal tags must not disrupt mitochondrial import. • Catalytically-dead rescue: the H158Y mutation abolishes desuccinylase/demalonylase activity and is the standard specificity control. • Mitochondrial localization validation: confirm mitochondrial localization by TOM20 co-staining or mitochondrial fractionation before functional assays. • Functional readout: rescue should restore desuccinylation of SIRT5 substrates measured by anti-succinyl-lysine Western blot. HEK293 transduces efficiently with lentivirus and supports stable rescue line generation.