The IFT25 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population that targets the IFT25 gene in the human HAP1 cell line. This gene disruption model provides a powerful loss-of-function tool for dissecting IFT25-dependent processes in ciliary biology and signal transduction. As a polyclonal pool, the cells represent a heterogeneous population of knockout alleles, enabling robust functional studies without clonal selection artifacts. This product is designed for researchers investigating intraflagellar transport, ciliogenesis, and associated signaling networks.
The HAP1 cell line is a near-haploid human cell line derived from a male patient with chronic myeloid leukemia. These adherent cells exhibit a fibroblast-like morphology and contain a single copy of most chromosomes, which simplifies genetic manipulation and phenotypic analysis. The near-haploid karyotype eliminates concerns about second-allele compensation, making HAP1 an ideal host for knockout studies. Its rapid growth and ease of culture further facilitate high-throughput screening and detailed biochemical assays.
IFT25 encodes an essential subunit of the intraflagellar transport complex B (IFT-B), which mediates anterograde transport of ciliary cargo along axonemal microtubules. IFT25 interacts with multiple IFT-B components, including IFT20, IFT27, IFT46, IFT52, IFT74, IFT81, and the kinesin-2 motor complex (KIF3A/KIF3B/KAP). Disruption of IFT25 prevents ciliary assembly, thereby blocking the Hedgehog signaling pathway. In the presence of Hedgehog ligands (SHH, IHH, DHH), the receptor Patched1 (PTCH1) normally relieves inhibition of Smoothened (SMO), allowing SMO to translocate into the cilium and activate GLI transcription factors. IFT25 knockout abrogates this cascade, resulting in failure to activate GLI1 and GLI2, and consequent downregulation of target genes such as CCND1, PTCH1, and HHIP. IFT25 also interfaces with Wnt signaling components, expanding its regulatory scope.
In HAP1 cells, loss of IFT25 produces a complete block in ciliogenesis, providing a clean genetic background to study cilium-dependent signaling. The near-haploid nature ensures that disruption of the single IFT25 allele yields a homogeneous loss-of-function phenotype across the population. This model is particularly valuable for dissecting Hedgehog pathway dynamics, as HAP1 cells retain functional GPCR signaling cascades. Researchers can use these cells to screen for small molecules that bypass IFT25 requirement or restore ciliary function, offering translational insights for ciliopathies such as retinal degeneration, skeletal dysplasias, and craniofacial abnormalities.
Typical applications include immunofluorescence-based ciliary length quantification using markers like acetylated ??-tubulin and ARL13B, western blotting for IFT-B complex integrity, and RT-qPCR profiling of GLI target transcripts. Hedgehog pathway activity can be monitored via Gli-luciferase reporter assays, while co-immunoprecipitation experiments can assess IFT25 interaction networks. The polyclonal format is well-suited for functional genomics screens, drug response profiling, and synthetic lethality studies. For further technical details and custom assay support, please contact Ascent Research.
