The BOLA2 Knockout HEK293T Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population derived from the widely used HEK293T host cell line, designed to achieve loss-of-function disruption of the BOLA2 gene. This product provides a genomically heterogeneous pool of edited cells, enabling researchers to interrogate BOLA2-dependent phenotypes in a population context that captures the spectrum of CRISPR-induced mutations. As a polyclonal population, it avoids clonal selection biases and is ideally suited for pooled functional assays, dose-response studies, and high-throughput screens where consistent gene disruption across replicates is required. The knockout model eliminates the need for in-house editing workflows, offering a ready-to-use system for studying iron-sulfur cluster metabolism and associated pathologies.
The host cell line, HEK293T, is an immortalized human embryonic kidney cell line that stably expresses the SV40 large T antigen, derived from the parental HEK293 cells. Its high transfectability and robust protein expression capacity make it a premier platform for recombinant protein production, gene editing feasibility, and mechanistic studies of cytosolic pathways. The embryonic kidney origin retains key metabolic and regulatory pathways relevant to iron homeostasis, providing a physiologically meaningful background for investigating BOLA2 function. Additionally, the SV40 large T antigen enhances episomal replication and allows for efficient amplification of vectors, facilitating downstream applications such as complementation rescue or overexpression studies to validate genotype-phenotype relationships.
BOLA2 is a core component of the cytosolic iron-sulfur cluster assembly (CIA) machinery, wherein it functionally partners with GLRX5 to mediate the transfer of pre-assembled Fe-S clusters onto recipient apoproteins. Its activity is regulated upstream by the NRF2 transcription factor, iron regulatory proteins, and cellular iron levels, while downstream targets include IRP1/cytosolic aconitase and other Fe-S cluster-dependent enzymes involved in metabolism, DNA repair, and gene expression. BOLA2 forms complexes with BOLA1, CIAO1, MMS19, and FAM96B, facilitating cluster delivery to targets via the CIA pathway. Interplay with additional pathway components such as NUBPL, ISCA1, and ISCA2 underscores its central role in maintaining cellular Fe-S protein maturation and overall iron homeostasis.
In the HEK293T context, BOLA2 knockout reveals critical insights into CIA pathway dynamics and iron-sulfur cluster-dependent regulation. This model is particularly relevant for studying 16p11.2 deletion syndrome, a genomic disorder that encompasses BOLA2 deletion and is associated with neurodevelopmental conditions including autism spectrum disorder and schizophrenia. The knockout cells also enable dissection of disordered iron-sulfur cluster metabolism, linking mitochondrial Fe-S export defects to cytosolic enzyme dysfunction. Moreover, cancer metabolism research benefits from this model, as many tumors exhibit heightened Fe-S cluster demand; the cells can be employed to screen for synthetic lethal interactions or drug sensitivities under iron-depleted conditions.
Research applications for these polyclonal knockout cells are extensive and include western blotting to confirm BOLA2 protein loss, RT-qPCR for transcript-level analysis, and aconitase activity assays to directly measure IRP1 function. Co-immunoprecipitation experiments can validate interactions with GLRX5, CIAO1, or MMS19, while immunofluorescence localizes any residual or mislocalized protein. Functional studies may incorporate cell proliferation assays under iron depletion, flow cytometry-based apoptosis detection following oxidative stress, and global transcriptomic analysis via RNA-seq to identify downstream effectors. These tools collectively support investigations into iron-sulfur cluster biogenesis, metal homeostasis, and disease modeling. For detailed protocols, assay conditions, and technical consultation, contact Ascent Research.
