Description

The NF1 Knockout U-251MG Cell Line is a CRISPR/Cas9-edited knockout cell line derived from the human U-251MG glioblastoma cell line. This engineered model carries a targeted disruption of the NF1 tumor suppressor gene, resulting in loss of functional neurofibromin protein. It provides a defined loss-of-function system for investigating NF1-dependent signaling and its role in glioblastoma tumorigenesis.

The parental U-251MG line is a widely used human glioblastoma model of the aggressive mesenchymal subtype. It features wild-type TP53, a loss-of-function mutation in the PTEN tumor suppressor, and amplification of the EGFR gene. These genetic lesions drive constitutive PI3K-AKT-mTOR pathway activity and enhanced receptor tyrosine kinase signaling, establishing a clinically relevant background for studying cooperative oncogenic effects with NF1 loss.

Neurofibromin functions as a GTPase-activating protein (GAP) for RAS family GTPases, including HRAS, KRAS, and NRAS. It accelerates the hydrolysis of active RAS-GTP to inactive RAS-GDP, thereby negatively regulating downstream effectors. Loss of NF1 GAP activity results in sustained RAS-GTP loading, which constitutively activates the RAF-MEK-ERK (BRAF/RAF1 ?? MEK1/2 ?? ERK1/2) and PI3K-AKT-mTOR signaling cascades. Additionally, RAS-dependent RalGEF?CRalA/B and transcription factors ELK1 and c-FOS are aberrantly engaged. The SPRED1 adaptor protein facilitates neurofibromin recruitment to the plasma membrane, and cAMP-PKA signaling modulates its activity.

In the context of U-251MG cells, NF1 knockout combines with pre-existing PTEN loss and EGFR amplification to hyperactivate both the MAPK and PI3K pathways, recapitulating the molecular features of NF1-mutant gliomas. This multi-hit model is especially suited for dissecting signaling network rewiring, adaptive resistance, and feedback mechanisms. It also provides a platform for identification of synthetic lethal interactions and evaluation of rational combination therapies targeting these oncogenic hubs.

This cell line supports a wide range of functional and pharmacological studies. Typical applications include dose-response screening of MEK inhibitors (such as trametinib) or PI3K/mTOR inhibitors, either alone or in combination. Cellular outcomes can be assessed via proliferation (MTT, BrdU, colony formation), apoptosis (Annexin V/PI staining), and migration/invasion (Transwell assays). Pathway activity is measurable through RAS-GTP pull-downs and western blotting for phospho-ERK1/2 and phospho-AKT. Gene expression changes can be quantified by RT-qPCR, and high-content assays such as phospho-kinase arrays are also compatible. The NF1 Knockout U-251MG Cell Line is a robust resource for glioblastoma research and therapeutic discovery. For technical inquiries and product information, please contact Ascent Research.