FLVCR2 Knockout HuH-7 Cell Line

Important nomenclature clarification: FLVCR2 (feline leukaemia virus subgroup C cellular receptor family member 2, MFSD7C, SLC49A2) was historically annotated as a heme transporter based on its sequence relationship to FLVCR1 and its identification as the feline FeLV-C receptor. However, recent breakthrough studies (Nguyen et al. Cell Research 2024; Nature 2024 structural studies) have established that the principal physiological substrate of human FLVCR2 is choline, not heme — FLVCR2 functions as a blood-brain barrier choline transporter responsible for approximately 60% of choline uptake into the brain. The choice between knockout and overexpression depends on whether you are studying FLVCR2 as a choline transporter or modeling Fowler syndrome. The Knockout line is the standard tool for asking whether FLVCR2 is required for choline transport or for FLVCR2-dependent processes. For BBB and choline transport research, the EDITGENE FLVCR2 Knockout in Huh-7 enables study of FLVCR2 biology — though physiological BBB choline transport requires brain microvascular endothelial cell models. Biallelic FLVCR2 loss-of-function mutations cause Fowler syndrome (proliferative vasculopathy and hydranencephaly-hydrocephaly, PVHH; OMIM 225790) — embryonic lethal cerebral vascular disease. Rescue with wild-type or transport-deficient FLVCR2 enables structure-function studies. The knockout is valuable for studying recently characterized FLVCR2 choline transport biology, Fowler syndrome mechanisms, and emerging brain choline delivery therapeutic strategies.

Yes. FLVCR2 rescue experiments require attention to MFS transporter architecture and the recently characterized choline transport function: • Construct design: use a codon-modified FLVCR2 sequence with a small intracellular tag (FLAG, HA). FLVCR2 has 12 transmembrane domains characteristic of MFS transporters — preserve membrane topology. • Surface localization validation: confirm plasma membrane localization by cell surface staining before choline transport assays. • Transport-deficient rescue: substrate-binding pocket mutations (e.g., aromatic and polar residues in the central choline-binding cavity, recently characterized by cryo-EM 2024) abolish choline transport and serve as the standard specificity control. • Fowler syndrome mutation rescue: patient-derived FLVCR2 mutations enable disease genotype-function studies. • Functional readout: rescue should restore choline uptake measured by ¹⁴C-choline or fluorescent choline analog cellular uptake. Huh-7 transduces efficiently with lentivirus and supports stable rescue line generation.