PPIF Knockout THLE-2 Cell Line

The choice depends on whether you are studying PPIF (cyclophilin D, CypD)'s role as a regulator of the mitochondrial permeability transition pore (mPTP) in human hepatic context or its functions in ischemia-reperfusion injury and metabolic stress. The Knockout line is the standard tool for asking whether CypD is required for mPTP opening in human hepatocytes — CypD sensitizes mPTP opening in response to mitochondrial Ca²⁺ overload, oxidative stress, and necrotic stimuli. Overexpression is useful for testing CypD effects on mitochondrial calcium handling. For human hepatic mitochondrial research, the EDITGENE PPIF Knockout in THLE-2 is highly relevant — THLE-2 is an SV40-immortalized normal human liver epithelial cell line, providing a human-relevant background for CypD-mPTP biology distinct from the parallel Ppif Knockout in mouse AML12 (also available). Rescue with wild-type or cyclosporin A-resistant CypD enables mechanistic studies. The knockout is a critical specificity control for cyclosporin A, NIM811, and alisporivir in hepatic ischemia-reperfusion and drug-induced liver injury research.

Primary applications: • Human hepatic mPTP opening: calcein-cobalt retention or membrane potential assessment following Ca²⁺ challenge to assess CypD-mPTP regulation in human hepatocyte context. • Hepatic ischemia-reperfusion modeling: anoxia/reoxygenation and oxidative stress assays in THLE-2's normal human liver epithelial background. • Drug-induced liver injury: acetaminophen, valproate, and other hepatotoxin sensitivity studies in CypD-null human hepatocytes. • mPTP inhibitor specificity: critical genetic control for cyclosporin A, NIM811, alisporivir in human-relevant hepatic protection research. EDITGENE recommends this human hepatic model for hepatic mitochondrial permeability transition research; the parallel Ppif Knockout in mouse AML12 (also available) complements for species-comparative studies.

Yes. CypD rescue experiments require attention to mitochondrial targeting: • Construct design: use a codon-modified PPIF sequence with a small C-terminal tag (FLAG, HA). CypD has N-terminal mitochondrial targeting sequence — N-terminal tags must not disrupt processing. • Mitochondrial localization validation: confirm mitochondrial matrix localization before functional assays. • Cyclosporin A-resistant rescue: specific mutations in the cyclosporin A binding pocket enable testing CsA on-target effects. • Functional readout: rescue should restore mPTP opening sensitivity to Ca²⁺ challenge measured by mitochondrial swelling, membrane potential loss, or calcein retention assays. THLE-2 is an SV40-immortalized normal human liver epithelial cell line — transduction with lentivirus is supported but may require optimization compared to standard transformed cell lines.