The IFT140 Knockout HAP1 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal population carrying disruptive mutations at the IFT140 locus in a near-haploid human cell background. This pooled knockout model provides a versatile loss-of-function system for studying ciliary biology and related signaling pathways without the clonal bias of single-cell isolates. The polyclonal format captures a spectrum of editing outcomes, making it suitable for both pooled screens and arrayed functional assays.
HAP1 cells are derived from the KBM-7 chronic myeloid leukemia line and maintain a haploid karyotype with an adherent, fibroblast-like morphology. The haploid state allows single-allele disruption to produce a functional null phenotype, simplifying loss-of-function analyses and enabling robust genetic screens. Widely used in functional genomics, HAP1 cells offer rapid growth, ease of transfection, and compatibility with diverse downstream detection methods.
IFT140 encodes a core subunit of the intraflagellar transport A (IFT-A) complex, which mediates retrograde trafficking of ciliary cargo from the tip to the cell body. IFT140 directly interacts with IFT-A partners IFT122, IFT144, and IFT139, and with the dynein-2 motor component DYNC2H1. Its expression is regulated by ciliogenic transcription factors RFX1, RFX2, RFX3, and FOXJ1. Loss of IFT140 abrogates retrograde IFT, causing defective ciliogenesis and deregulation of cilium-dependent signaling. In the Hedgehog pathway, IFT140 knockout impairs processing of GLI1, GLI2, and GLI3 downstream of Smoothened (SMO), disrupting both activator and repressor functions. Concurrently, Wnt/planar cell polarity signaling is compromised, affecting ??-catenin, Dishevelled (DVL), and Vangl2-dependent outputs.
In the HAP1 context, IFT140 knockout leads to severely shortened or absent primary cilia, readily visualized by immunofluorescence for ARL13B and acetylated ??-tubulin. The haploid background ensures a penetrant phenotype and facilitates complementation by wild-type IFT140 re-expression. These cells exhibit aberrant Hedgehog and Wnt pathway activity, as measurable by RT-qPCR of target genes like GLI1 and PTCH1, providing a clean genetic platform to dissect IFT-A-dependent trafficking and signaling.
Key applications include modeling ciliopathies such as short-rib thoracic dysplasias (Jeune syndrome, Mainzer-Saldino syndrome), screening for modifiers of ciliogenesis, and mechanistic dissection of IFT-A complex function. Researchers can employ the cells in ciliogenesis assays, co-immunoprecipitation of IFT components, and transcriptomic profiling to map IFT140-dependent gene networks. The polyclonal pool is compatible with high-content imaging, flow cytometry for cilia frequency, and pharmacological modulation of Hedgehog or Wnt pathways. For additional information, please contact Ascent Research.
