The HSD3B1 Knockout HAP1 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout population designed to ablate expression of the 3??-hydroxysteroid dehydrogenase/??5-4 isomerase type 1 (HSD3B1) gene in the HAP1 near-haploid human cell line. This product provides a polyclonal gene disruption model, enabling loss-of-function studies without reliance on single-cell clonal isolation. The polyclonal format maintains genetic heterogeneity while targeting the entire coding sequence of HSD3B1, offering a robust platform for functional genomics, steroid signaling research, and drug discovery applications.
HAP1 is a near-haploid cell line derived from the male KBM-7 chronic myeloid leukemia line, characterized by a single copy of most chromosomes except for a disomic region of chromosome 15. Notably, HAP1 cells lack expression of several HLA class I genes, reducing immunogenicity artifacts in high-throughput screening. Their haploid genetics facilitate unambiguous gene disruption, making HAP1 a widely adopted model for CRISPR-based functional genomic screens, pathway dissection, and synthetic biology across a range of research areas including cancer, metabolism, and signal transduction.
HSD3B1 encodes the enzyme responsible for the oxidative conversion of ??5-3??-hydroxysteroid precursors??such as pregnenolone, 17??-hydroxypregnenolone, and dehydroepiandrosterone (DHEA)??into their corresponding ??4-3-ketosteroids, including progesterone, 17??-hydroxyprogesterone, and androstenedione. This reaction is a critical step in the biosynthesis of all major steroid classes: progestins, glucocorticoids, mineralocorticoids, androgens, and estrogens. Transcription of HSD3B1 is regulated upstream by NR5A1 (SF-1), GATA4, and cAMP/PKA signaling pathways, which are activated by trophic hormones such as luteinizing hormone (LH)/human chorionic gonadotropin (hCG), angiotensin II, and adrenocorticotropic hormone (ACTH). HSD3B1 functions in close collaboration with cytochrome P450 enzymes CYP17A1 and CYP21A2, and its catalytic activity requires NAD+ and membrane phospholipids. Downstream, HSD3B1-generated ??4-3-ketosteroids serve as substrates for further conversion to cortisol, aldosterone, testosterone, and estradiol, which then exert biological effects through androgen, progesterone, and estrogen receptors.
In the context of HAP1 cells, disruption of HSD3B1 eliminates the capacity to produce progesterone and its downstream steroid metabolites, thereby creating a clean genetic background for interrogating steroid-dependent signaling pathways. Although HAP1 is a leukemic myeloid line, its near-haploid genome simplifies the analysis of steroidogenic gene function, allowing researchers to bypass compensatory isoenzyme activity that can complicate studies in diploid cells. This model is particularly valuable for dissecting the role of HSD3B1 in castration-resistant prostate cancer, where increased enzyme expression promotes intratumoral androgen synthesis from adrenal precursors, and for investigating congenital adrenal hyperplasia-associated mutations. The polyclonal population also supports metabolic engineering efforts aimed at re-routing steroid flux in heterologous expression systems.
Typical research applications include functional genomics of steroidogenesis, CRISPR knockout screening for steroid pathway dependencies, drug target validation for castration-resistant prostate cancer, metabolic engineering of steroid hormone production, and synthetic lethality screens in steroidogenic contexts. Representative assays compatible with this model encompass Western blotting and RT-qPCR for gene expression validation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) for steroid hormone profiling, androgen receptor reporter assays, immunofluorescence, RNA-seq transcriptomics, flow cytometry for cell cycle effects, and phospho-signaling analysis of AKT/ERK pathways. For additional technical specifications, please contact Ascent Research.
