BOLA3 Knockout HAP1 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal population of near-haploid HAP1 cells in which the BOLA3 gene has been disrupted, providing a loss-of-function model for mitochondrial iron-sulfur (Fe-S) cluster assembly research. The polyclonal format captures a heterogeneous allele spectrum, avoiding clonal artifacts and enabling robust population-level analyses. This product is suitable for investigating BOLA3-dependent pathways without the constraints of single-cell cloning.
The HAP1 cell line is a human chronic myeloid leukemia (CML)-derived, fibroblast-like line with a stable near-haploid karyotype, making it ideal for genetic screens and knockout studies. Single-copy gene disruptions yield immediate phenotypic outcomes, eliminating the complexity of diploid genetics. The HAP1 background has been well-characterized for mitochondrial function, offering a reliable platform for studying BOLA3-related metabolic and respiratory defects.
BOLA3 functions in the late steps of mitochondrial [4Fe-4S] cluster transfer, interacting with NFU1, GLRX5, ISCA1, and ISCA2 to deliver clusters to downstream targets such as lipoic acid synthase (LIAS) and respiratory chain complex I subunits. Upstream regulation involves mitochondrial biogenesis and iron availability, while downstream effects include maturation of ACO2 and LIAS. BOLA3 knockout disrupts key Fe-S dependent processes, impairing lipoic acid biosynthesis and oxidative phosphorylation.
In HAP1 cells, BOLA3 knockout recapitulates features of multiple mitochondrial dysfunctions syndrome type 2 (MMDS2), characterized by defective Fe-S cluster maturation. The haploid genetic background simplifies dissection of mitochondrial phenotypes, such as reduced respiratory chain activity and altered iron homeostasis. This model enables examination of BOLA3’s role in cellular energetics and lipoic acid utilization, facilitating studies of disease mechanisms and potential therapeutic interventions.
Applications include mechanistic studies of Fe-S cluster biogenesis, disease modeling of MMDS2, functional genomics screening, and pharmacological rescue experiments. Assays frequently employed are Western blotting of LIAS and complex I subunits, RT-qPCR for mitochondrial stress genes, RNA-seq transcriptomics, flow cytometry for mitochondrial membrane potential, Seahorse metabolic flux analysis, and co-immunoprecipitation of BOLA3 with NFU1/GLRX5. For further technical details, please contact Ascent Research.
