MFSD5 Knockout HCT116 Cell Line

The choice depends on whether you are studying SLC61A1 (MFSD5/hsMOT2)'s role as a molybdate transporter or its emerging functions in cellular molybdenum homeostasis. Note that SLC61A1 is the current HGNC-approved symbol; this gene is also widely referred to as MFSD5 or hsMOT2 in published literature, and is characterized as a high-affinity intracellular molybdate transporter, the human homolog of the algal MoT2 transporter. The Knockout line is appropriate for asking whether SLC61A1 is required for cellular molybdate uptake and downstream molybdopterin-dependent enzyme activities — particularly relevant given that molybdenum is an essential cofactor for sulfite oxidase, aldehyde oxidase, and xanthine dehydrogenase. Overexpression is useful for testing transport activity in heterologous systems or for studying substrate specificity. For molybdenum biology research, the EDITGENE SLC61A1 Knockout in HCT 116 enables study of molybdate-dependent enzymatic activities and the cellular consequences of disrupted molybdenum cofactor (Moco) biosynthesis in a colorectal cancer background. Rescue with wild-type or transport-deficient SLC61A1 is the standard specificity control for distinguishing transport activity from non-transport functions.

Primary applications: • Molybdate uptake assays: cellular uptake of ⁹⁹Mo-labeled molybdate or LC-ICP-MS-based intracellular molybdenum measurement to quantify SLC61A1 transport activity. • Molybdoenzyme activity: sulfite oxidase, xanthine oxidase, and aldehyde oxidase activity assays to assess downstream consequences of disrupted molybdate uptake on Moco-dependent enzymes. • Molybdenum cofactor (Moco) biosynthesis: analysis of pterin intermediates and Moco-dependent processes in the absence of SLC61A1. • Heterologous transport characterization: rescue with wild-type SLC61A1 in yeast or HEK293 heterologous systems, complementing transport activity studies in the colorectal cancer HCT 116 background. EDITGENE recommends this model for researchers investigating molybdate transport biology, molybdenum cofactor metabolism, and Moco-dependent enzyme biology.

Yes. SLC61A1 rescue experiments require attention to membrane topology and transport mechanism: • Construct design: use a codon-modified SLC61A1 sequence with a small C-terminal tag (FLAG, HA). SLC61A1 is a 450-amino-acid MFS-family transporter with 12 predicted transmembrane domains — N-terminal tags must not disrupt membrane topology. • Transport-deficient rescue: substrate-binding pocket mutations or conserved MFS family residue mutations enable distinguishing transport activity from non-transport functions; the algal MoT2 homolog provides a reference for structure-function-relevant residue identification. • Surface and intracellular localization validation: SLC61A1 has been reported with both plasma membrane and intracellular membrane localization — confirm exogenous protein localization by imaging or fractionation before functional assays. • Functional readout: rescue should restore cellular molybdate uptake (measured by ICP-MS) and downstream molybdoenzyme activities (e.g., sulfite oxidase activity). HCT 116 transduces efficiently with lentivirus and supports stable rescue line generation; the colorectal cancer background may be relevant given emerging connections between micronutrient transport and cancer metabolism.