The KIFAP3 Knockout HAP1 Polyclonal Cells product provides a CRISPR/Cas9-edited polyclonal knockout cell population targeting the KIFAP3 gene in HAP1 cells. This gene-edited pool is designed for functional studies requiring loss of KIFAP3 expression without clonal isolation, enabling robust analysis of gene function in a controlled genetic background. The polyclonal nature of the knockout population ensures genetic diversity while maintaining consistent disruption of the target locus, making it suitable for high-throughput screening and pathway interrogation in cell-based assays.
HAP1 is a near-haploid human cell line derived from the KBM-7 chronic myeloid leukemia isolate. Its haploid karyotype simplifies gene knockout studies, as a single targeting event is sufficient to disrupt gene function, facilitating the generation of homozygous-like null phenotypes. HAP1 cells retain the capacity to form primary cilia, express key components of the hedgehog signaling machinery, and are amenable to a wide range of molecular and imaging assays, positioning them as a versatile model system for studying ciliary biology and signal transduction.
KIFAP3 encodes a non-motor subunit of the heterotrimeric kinesin-2 motor complex, which is essential for anterograde intraflagellar transport (IFT) along ciliary microtubules. KIFAP3 forms a functional complex with motor subunits KIF3A and KIF3B and interacts with IFT particles including IFT20 and IFT88 to mediate cargo delivery required for ciliogenesis. Its activity is modulated by phosphorylation via upstream kinases CDK5 and GSK3??. Disruption of KIFAP3 abolishes proper IFT, leading to defective ciliary assembly and attenuated hedgehog signaling. This is evidenced by impaired translocation of smoothened (SMO) and reduced transcriptional activity of GLI2, a downstream effector, ultimately compromising the expression of hedgehog target genes such as GLI1.
In the HAP1 cellular context, KIFAP3 knockout disrupts the formation and maintenance of primary cilia, providing a powerful model to dissect the molecular requirements of IFT-dependent ciliogenesis. Because HAP1 cells rely on functional kinesin-2 for hedgehog signal transduction, loss of KIFAP3 results in blockade of the pathway, mimicking aspects of ciliopathy phenotypes. This isogenic system allows direct comparison between wild-type and KIFAP3-deficient cells, enabling quantitative assessment of ciliary length, trafficking dynamics, and signal responsiveness without confounding genetic variability. The model is therefore particularly relevant for investigating mechanisms underlying retinitis pigmentosa and other ciliopathies where kinesin-2 dysfunction is implicated.
Typical applications include ciliopathy modeling, hedgehog pathway dissection, intracellular transport studies, and validation of CRISPR screening hits. Researchers can employ western blotting to confirm KIFAP3 protein depletion, immunofluorescence to visualize ciliary markers such as IFT88 and ARL13B, RT-qPCR to measure GLI1 and GLI2 transcript levels, and cilia formation assays to quantify ciliogenesis defects. Flow cytometry-based ciliary signaling assays and hedgehog luciferase reporter systems further extend the utility of this model in drug discovery and functional genomics. For further technical details or to place an order, please contact Ascent Research.
