The DIO3 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population targeting the human DIO3 gene in HAP1 cells. This heterogeneous pool provides a convenient loss-of-function model for studying type 3 deiodinase activity without requiring single-cell cloning.
HAP1 cells are a near-haploid, adherent human cell line derived from the chronic myeloid leukemia line KBM-7. Their haploid genome simplifies genetic knockout studies by enabling clear phenotypic readouts from single-copy gene disruptions. HAP1 cells are widely used for functional genomics, signaling pathway analysis, and drug target validation.
DIO3 encodes a selenocysteine-dependent inner ring deiodinase that inactivates thyroid hormones by converting the prohormone thyroxine (T4) to reverse triiodothyronine (rT3) and active triiodothyronine (T3) to inactive 3,3??-diiodothyronine (T2). This reduces intracellular T3 availability, thereby suppressing thyroid hormone receptor (THRA/THRB)-mediated transcription. THRs form heterodimers with retinoid X receptors (RXRs) to regulate gene expression. DIO3??s activity is controlled by upstream signals including thyroid hormones themselves, the transcription factor FoxO1, Hedgehog signaling, TGF-??, and hypoxia, and it requires selenocysteine incorporation machinery and interaction with thyroid hormone transporters such as MCT8 (SLC16A2) for substrate access. In contrast, related deiodinases DIO1 and DIO2 catalyze activating outer ring deiodination. Downstream, DIO3 action diminishes expression of T3-responsive genes, suppresses metabolic rate, influences developmental processes, and modulates pathways such as Wnt/??-catenin.
In the HAP1 near-haploid background, CRISPR/Cas9-mediated DIO3 disruption leads to loss of thyroid hormone inactivation, elevating intracellular T3 and constitutively activating T3-responsive transcriptional programs. This unmasks the gene??s roles in regulating cell proliferation and differentiation, making the model relevant for dissecting its contributions to thyroid disorders, metabolic syndrome, and tumorigenesis??contexts in which aberrant DIO3 expression has been implicated, including hepatocellular carcinoma and breast cancer. The polyclonal knockout population offers a uniform loss-of-function phenotype due to haploidy, facilitating robust and reproducible downstream assays.
This DIO3 knockout cell pool supports diverse experimental workflows: gene expression analysis by RT-qPCR, protein detection by Western blot, cellular thyroid hormone quantification via ELISA or LC-MS, proliferation and metabolic flux assays, transcriptome-wide RNA-seq, and luciferase reporter assays driven by thyroid hormone response elements. Researchers can apply the model to study thyroid hormone metabolism, screen compounds targeting endocrine pathways, investigate developmental signaling, and perform genetic interaction screens in a clean haploid system. For additional information or customization, contact Ascent Research.
