The AKR1D1 Knockout HAP1 Polyclonal Cells product is a CRISPR/Cas9-edited polyclonal knockout cell population in the HAP1 background, providing targeted disruption of the human AKR1D1 gene. This cell pool is engineered for loss-of-function studies in bile acid biosynthesis and steroid hormone metabolism. The polyclonal format captures a diverse range of editing events, facilitating robust functional analyses without clonal bias. The AKR1D1 locus has been inactivated via CRISPR/Cas9-mediated gene disruption, creating a versatile model for genetic and pharmacological investigations.
HAP1 is a near-haploid human cell line derived from the KBM-7 chronic myeloid leukemia line. These male-derived cells exhibit adherent fibroblast-like morphology and a predominantly haploid genome, allowing unambiguous genotype-phenotype correlation. The near-haploid state simplifies genetic manipulation and makes HAP1 an ideal host for functional genomics screens, particularly for recessive phenotypes. This characteristic enhances the utility of the AKR1D1 knockout, as single-allele disruption leads to clear functional deficiency without diploid compensation.
AKR1D1 encodes steroid 5-beta-reductase, an aldo-keto reductase that catalyzes NADPH-dependent 5??-reduction of ??4-3-ketosteroids. This reaction is a critical step in primary bile acid synthesis, functioning downstream of CYP7A1 and CYP8B1 to convert 7??-hydroxy-4-cholesten-3-one into 5??-reduced intermediates, which are further processed by CYP27A1. AKR1D1 is transcriptionally regulated by the nuclear receptor FXR (NR1H4) and HNF4alpha in response to bile acid levels, and it also inactivates steroid hormones. Loss of AKR1D1 function causes accumulation of toxic bile acid intermediates, leading to congenital bile acid synthesis defect type 2 (CBAS2) and cholestatic liver disease.
In the HAP1 background, AKR1D1 knockout provides a clear cellular model of bile acid and steroid metabolism disorders. The lack of a second allele ensures that any observed phenotype is directly attributable to AKR1D1 disruption, making it particularly informative for studying the enzyme??s role in substrate conversion and pathway regulation. This model enables detailed metabolic profiling and toxicity assays, while the haploid genome facilitates the identification of genetic modifiers of AKR1D1-related pathology through CRISPR screens.
This polyclonal knockout pool supports diverse applications such as LC-MS-based bile acid profiling, steroid conversion assays, and viability assessments under bile acid stress. Researchers can validate AKR1D1 inactivation via RT-qPCR and Western blot, and probe interacting partners like CYP7A1 and FXR. The model is valuable for investigating congenital cholestasis and drug metabolism. It is also suited for high-throughput genetic modifier screens to uncover synthetic lethal interactions or therapeutic targets. For further information, contact Ascent Research.
