ISYNA1 knockout HEK293T polyclonal cells are a CRISPR/Cas9-edited cell population with targeted disruption of the ISYNA1 gene. This genetically heterogeneous model enables loss-of-function studies of inositol metabolism and phosphatidylinositol signaling without the biases of single-cell clones. The polyclonal format mitigates off-target concerns and provides a robust platform for biochemical and cell-based assays in a scalable HEK293T background.
The HEK293T host line, derived from human embryonic kidney cells and transformed with SV40 large T-antigen, is widely used for viral production and protein overexpression. Its well-characterized metabolic profile and amenability to lipidomic and phosphoproteomic analyses make it suitable for studying metabolic enzymes like ISYNA1. Rapid proliferation further enhances its utility for knockout model generation.
ISYNA1 encodes inositol-3-phosphate synthase 1, catalyzing the rate-limiting conversion of glucose-6-phosphate to inositol-3-phosphate in de novo myo-inositol biosynthesis. Its activity is regulated by substrate availability, cellular energy status, and MYC transcription factor. Myo-inositol feeds phosphatidylinositol synthesis, leading to PI(3,4,5)P3 generation by PI3K and AKT activation. The enzyme interacts with IMPA1 and SMIT1/2 transporters. Knockout disrupts myo-inositol production, thereby impairing phosphatidylinositol, PI(4,5)P2, PI(3,4,5)P3, and IP3 synthesis, attenuating PI3K/AKT signaling.
In HEK293T cells, ISYNA1 knockout forces dependence on exogenous myo-inositol from medium, enabling study of intracellular myo-inositol homeostasis and the balance between synthesis and uptake. This model is valuable for investigating neural tube defects and inositol metabolism disorders, as ISYNA1 mutations link to spina bifida. The polyclonal pool allows exploration of metabolic adaptation under inositol limitation.
These cells support dissection of myo-inositol roles in PI3K/AKT signaling via phospho-AKT western blot, proliferation assays, and Annexin V apoptosis staining under varying inositol. Metabolite quantification by LC-MS and ISYNA1 expression analysis via RT-qPCR and western blot confirm knockout. Applications include metabolic engineering, neurodevelopment modeling, and high-throughput screening for inositol pathway modulators. For more information, contact Ascent Research.
