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The U-2 OS-CRISPR-NUP96-SNAP is a genetically modified osteosarcoma cell line derived from the parent U-2 OS human cell line. This cell line has been engineered through CRISPR/Cas9-mediated genome editing to incorporate a SNAP-tag at the NUP96 gene, enabling the visualization and study of nuclear pore complex dynamics. Nuclear pore complexes (NPCs) are crucial for the regulation of nucleocytoplasmic transport, and NUP96 is a significant component of the NPC, playing a pivotal role in its structural integrity and function.
In U-2 OS-CRISPR-NUP96-SNAP clone no.33, the integration of the SNAP-tag at the NUP96 locus allows for specific and covalent attachment of fluorescent substrates or other chemical probes that can be used for live-cell imaging and other biochemical assays. This feature makes it an invaluable tool for investigating the molecular dynamics of nucleocytoplasmic transport, understanding NPC-related pathologies, and screening for therapeutic compounds that affect NPC function. The cell line also retains the characteristics of the parental U-2 OS line, which includes a high level of genetic stability and ease of culture, making it suitable for high-throughput screening and extended studies in cell biology.
Due to the specificity of the modification at the NUP96 gene, U-2 OS-CRISPR-NUP96-SNAP clone no.33 provides a unique model for the detailed study of NPC components in the context of cellular function and dysfunction. Researchers can exploit the SNAP-tag system to selectively and rapidly label NUP96, facilitating real-time visualization of NPC dynamics under physiological and pathological conditions. This specific clone can serve as a robust platform for both basic research and applied biomedical studies, contributing significantly to the fields of cellular biology, genetics, and oncology.
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Protein expression: NUP96-SNAP (nuclear pore complex protein 96, SNAP-tag)
- cultureMedium: McCoys 5a, w: 3.0 g/L Glucose, w: stable Glutamine, w: 2.0 mM Sodium pyruvate, w: 2.2 g/L NaHCO3 (Cytion article number 820200a)
- supplements: Supplement the medium with 10% FBS, 3.0 g/L Glucose, stable Glutamine, 2.0 mM Sodium pyruvate, 2.2 g/L NaHCO3, 1% NEAA
- dissociationReagent: Accutase
- subculturing: Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium.
- seedingDensity: 1 x 104 cells/cm2
- fluidRenewal: 2 to 3 times per week
- freezeMedium: As a cryopreservation medium, use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress.
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