U2OS-CRISPR-SNAPf-Nup133

U2OS-CRISPR-SNAPf-Nup133 is a genetically engineered human osteosarcoma cell line derived from the parental U2OS background in which the endogenous NUP133 locus has been modified using CRISPR/Cas9-mediated genome editing to encode a C-terminal SNAPf tag. NUP133 is a core component of the Y-complex (NUP107-160 complex), a structural subcomplex essential for nuclear pore complex (NPC) assembly and maintenance. By introducing the SNAPf coding sequence in-frame at the endogenous locus, the fusion protein is expressed under native regulatory control, preserving physiological expression levels and subcellular localization.

The SNAPf tag is a fast-labeling variant of the SNAP-tag, an engineered O6-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine-conjugated substrates. This enables highly specific and versatile fluorescent labeling of Nup133 in live or fixed cells using cell-permeable or impermeable SNAP substrates. In U2OS-CRISPR-SNAPf-Nup133 cells, the fusion protein localizes to the nuclear envelope in a punctate pattern characteristic of nuclear pore complexes. Because tagging occurs at the endogenous locus, NPC stoichiometry and architecture are minimally perturbed, making this model suitable for quantitative super-resolution microscopy, single-molecule tracking, and kinetic analyses of NPC assembly and turnover.

This cell line provides a robust platform for studying nuclear transport, nucleocytoplasmic trafficking dynamics, NPC biogenesis during interphase and post-mitotic nuclear reassembly, and structural organization of the Y-complex within the pore scaffold. The U2OS background offers flat morphology and large nuclei, facilitating high-resolution imaging. U2OS-CRISPR-SNAPf-Nup133 cells are particularly well suited for pulse-chase labeling experiments, correlative light and electron microscopy, and multicolor imaging approaches in combination with additional endogenously tagged nucleoporins or transport factors.

37°C, 5% CO₂, humidified atmosphere.

1. Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit.

2. Upon receipt, either store the cryovial immediately at temperatures below -150°C to ensure the preservation of cellular integrity, or proceed to step 3 if immediate culturing is required.

3. For immediate culturing, swiftly thaw the vial by immersing it in a 37°C water bath with clean water and an antimicrobial agent, agitating gently for 40-60 seconds until a small ice clump remains.

4. Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening.

5. Carefully open the disinfected vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of room-temperature culture medium, mixing gently.

6. Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium.

7. Gently resuspend the cell pellet in 10 ml of fresh culture medium. For adherent cells, divide the suspension between two T25 culture flasks; for suspension cultures, transfer all the medium into one T25 flask to promote effective cell interaction and growth.

8. Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes.

Mycoplasma contamination is excluded using both PCR-based assays and luminescence-based mycoplasma detection methods.

To ensure there is no bacterial, fungal, or yeast contamination, cell cultures are subjected to daily visual inspections.

For long-term preservation, place vials in vapor-phase liquid nitrogen at about −150 to −196 °C. Storage at −80 °C is acceptable only as a short interim step before transfer to liquid nitrogen.

Cryopreserved cell lines are shipped on dry ice in validated, insulated packaging with sufficient refrigerant to maintain approximately −78 °C throughout transit. On receipt, inspect the container immediately and transfer vials without delay to appropriate storage.

At Cytion, we stand behind the quality and viability of our products. We guarantee that our cells will be viable at the start of culture for 60 days after shipment from our facilities. We assure that all materials conform to the specifications described in the Certificate of Analysis and/or Product Sheet.

For full details, please refer to our Warranty Statement.