MFSD14A Knockout HCT116 Cell Line

The choice depends on whether you are studying SLC71A1 (MFSD14A/HIAT1)'s role as a Golgi-localized MFS-family transporter or its emerging functions in energy homeostasis and reproductive biology. Note that SLC71A1 is the current HGNC-approved symbol; this gene is widely referred to as MFSD14A or HIAT1 (hippocampus abundant transcript 1) in published literature. It shows sequence and structural homology with sugar transporters — including the GLUT2/GLUT4 glucose transporters and bacterial tetracycline resistance proteins — possessing the characteristic 12-transmembrane α-helical architecture and the P-E-S-P-R motif found in facilitative glucose transporters, although its physiological substrate remains uncharacterized. The Knockout line is appropriate for asking whether SLC71A1 is required for cellular phenotypes connected to its established biology — published Mfsd14a/Hiat1 knockout mice show globozoospermia and male infertility (Spermatogenic Failure 19), and nutrient-availability studies have shown SLC71A1 expression changes with amino acid starvation and feeding state. Overexpression in HCT 116 may inform on substrate identification and trafficking. For physiological function research, particularly reproductive biology, germ-cell models are more appropriate; for energy homeostasis, neuronal or hepatic models complement the HCT 116 background. Rescue with wild-type SLC71A1 is the standard specificity control.

Primary applications: • Substrate discovery: untargeted metabolomics in the knockout to identify candidate organic substrates by detecting altered intracellular metabolite levels — phylogenetic clustering predicts SLC71A1 has an organic substrate profile relevant to energy homeostasis. • Nutrient sensing studies: transcriptomic and protein-level analysis under amino acid starvation and refeeding conditions, given that Mfsd14a expression is upregulated by 3-hour amino acid starvation. • Subcellular localization studies: rescue with epitope-tagged SLC71A1 to confirm Golgi localization (Giantin co-staining) and characterize trafficking determinants in the HCT 116 background. • Disease variant studies: rescue with Spermatogenic Failure 19-associated SLC71A1 variants for genotype-function correlation studies in a tractable cellular context. EDITGENE recommends this model for in vitro biochemistry, substrate discovery, and disease variant studies. Reproductive function research requires germ-cell-derived models, and physiological energy homeostasis research benefits from neuronal or hepatic complementary models.

Yes. SLC71A1 rescue experiments require attention to MFS family transporter architecture: • Construct design: use a codon-modified SLC71A1 sequence with a small C-terminal tag (FLAG, HA). SLC71A1/MFSD14A is a 490-amino-acid MFS transporter with 12 predicted transmembrane domains — N-terminal tags must not disrupt membrane topology. • Golgi localization validation: confirm Golgi targeting of exogenous SLC71A1 by Giantin co-staining before functional assays; published evidence indicates Golgi rather than plasma membrane localization in neurons. • Transport-deficient rescue: mutations in conserved MFS family residues (e.g., the D-R/K-X-G-R-R/K motif between TM2 and TM3, or the P-E-S-P-R motif at the end of TM6) enable distinguishing transport activity from non-transport functions. • Disease variant rescue: introduction of Spermatogenic Failure 19-associated mutations enables genotype-function correlation studies in a controlled HCT 116 background. HCT 116 transduces efficiently with lentivirus and supports stable rescue line generation; the colorectal cancer background is appropriate for biochemical mechanism studies and substrate discovery experiments.