The KIF5A Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited cell population with targeted disruption of the KIF5A gene, which encodes the heavy chain of the kinesin-1 motor protein. This polyclonal knockout pool provides a loss-of-function model for studying KIF5A-dependent intracellular transport without the need for single-cell cloning. Generated by electroporation of ribonucleoprotein complexes, the heterogeneous population enables functional studies in a genetically diverse background that reflects population-level responses to KIF5A ablation.
The host HEK293T cell line, derived from human embryonic kidney epithelial cells, is widely used for high-level protein expression and viral production due to its exceptional transfection efficiency and stable expression of the SV40 large T-antigen, which allows episomal replication of plasmids containing the SV40 origin. These features make HEK293T ideal for transient introduction of reporters or rescue constructs alongside the knockout background, facilitating mechanistic dissection of kinesin-1 function in a tractable system.
KIF5A encodes the heavy chain subunit of the kinesin-1 motor complex, driving anterograde transport of cargoes including mitochondria, neurofilaments, and signaling endosomes along microtubules. KIF5A activity is regulated by JNK-mediated phosphorylation via JIP1 scaffold proteins and Ca2+/calmodulin signaling, and it interacts with cargo-specific adaptors such as TRAK1/2 (Milton) and HAP1. Downstream, KIF5A maintains mitochondrial distribution and neurofilament assembly, processes essential for neuronal homeostasis; its dysfunction is linked to hereditary spastic paraplegia type 10, Charcot-Marie-Tooth disease type 2, and amyotrophic lateral sclerosis.
Despite the non-neuronal origin of HEK293T cells, they express core microtubule-based transport machinery, enabling investigation of fundamental KIF5A motor functions in a simplified system. This knockout model is particularly suited for studying cargo loading, motor processivity, and adaptor interactions without neuronal isoform complexity. High transfection efficiency also supports complementation with wild-type or disease-mutant KIF5A constructs, facilitating genotype-phenotype correlations relevant to neurodegenerative disease research.
Research applications include modeling axonal transport deficits via live-cell imaging of organelle trafficking, screening modulators of kinesin activity, and probing protein interactions by co-immunoprecipitation. Functional assays such as JC-1 staining for mitochondrial membrane potential and MTT viability assays provide downstream readouts, while Western blotting and RT-qPCR confirm knockout efficiency. RNA-seq can reveal transcriptomic consequences of KIF5A loss. For inquiries or ordering, contact Ascent Research.
