The IFT74 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HT29 human colorectal adenocarcinoma cell line. This product enables targeted disruption of the IFT74 gene, which encodes a core subunit of the intraflagellar transport complex B (IFT-B). The polyclonal nature provides a heterogeneous pool of gene-disrupted cells that can be used to interrogate IFT74-dependent functions without clonal biases. No specific editing outcome is implied; cells contain a spectrum of CRISPR-mediated disruptions that impair IFT74 protein expression and downstream biological activities.
HT29 is a well-characterized adherent epithelial cell line established from a primary colon adenocarcinoma of a 44-year-old Caucasian female. It maintains a near-diploid karyotype and expresses typical colonocyte markers, making it a reliable model for colorectal cancer biology and intestinal epithelial physiology. HT29 cells exhibit active Wnt/??-catenin signaling due to APC mutation, and they form tumors in xenograft models. The combination of this genetic background with IFT74 disruption allows dissection of cilia-dependent and -independent signaling pathways relevant to colorectal tumorigenesis.
IFT74 functions as a backbone component of the IFT-B complex, essential for anterograde transport along the ciliary axoneme and primary cilium assembly. It interacts directly with IFT81 and forms complexes with other IFT-B members including IFT20, IFT52, and IFT88, as well as motors KIF3B and DYNC2H1. IFT74 transcription is regulated by ciliogenic transcription factors such as FOXJ1 and RFX3. Through its role in ciliogenesis, IFT74 modulates Hedgehog signaling: proper cilium structure is required for processing of GLI transcription factors downstream of SMO. Simultaneously, IFT74 influences Wnt/??-catenin signaling; in HT29 cells, disruption of IFT74 attenuates ??-catenin/TCF4 transcriptional activity, reducing expression of targets such as CCND1 (cyclin D1) and AXIN2. This molecular network connects ciliary integrity to cell cycle control and proliferation.
In the context of colorectal adenocarcinoma, IFT74 knockout provides a powerful loss-of-function model to study how primary cilium dysfunction intersects with oncogenic pathways. HT29 cells typically lack primary cilia due to culture conditions, yet IFT-B components may still exert non-ciliary functions; IFT74 depletion can reveal cilia-independent roles in Wnt pathway regulation and cell cycle progression. The model is particularly suited for investigating ciliopathy-related mechanisms in a cancer setting, including Bardet-Biedl syndrome gene interactions. Moreover, the interplay between IFT74, ??-catenin, and cyclin D1 offers a platform for evaluating therapeutic strategies that target Wnt/??-catenin-driven colorectal cancer.
Researchers can employ this polyclonal knockout population in a range of assays. Ciliary phenotypes are assessed by immunofluorescence staining for ciliary markers such as Arl13b and acetylated ??-tubulin. Western blotting confirms loss of IFT74 and monitors changes in ??-catenin and cyclin D1 levels. Transcriptional outputs are measured via RT-qPCR for GLI1 and AXIN2. Wnt signaling activity can be quantified using TOPFlash/FOPFlash reporter assays. Functional studies include MTS proliferation assays, flow cytometric cell cycle analysis, and scratch wound migration assays. The product is also applicable in drug sensitivity screens targeting Hedgehog or Wnt pathways. For further information, please contact Ascent Research.
