U2OS-CRISPR-SNAPf-Nup358/RanBP2

U2OS-CRISPR-SNAPf-Nup358/RanBP2 is a genome-edited human osteosarcoma cell line derived from U2OS cells in which the endogenous RANBP2 (also known as NUP358) locus has been modified by CRISPR/Cas9 to encode a SNAPf tag in-frame with the native protein. Nup358/RanBP2 is a large nucleoporin localized to the cytoplasmic filaments of the nuclear pore complex (NPC) and plays critical roles in nucleocytoplasmic transport, SUMOylation, and mitotic processes. Endogenous tagging ensures that SNAPf-Nup358 is expressed under physiological promoter control, maintaining native expression levels and minimizing artifacts associated with overexpression systems.

The SNAPf tag is a fast-labeling variant of the SNAP-tag that covalently binds benzylguanine-conjugated substrates, enabling selective and stable fluorescent labeling of Nup358 in live or fixed cells. In U2OS-CRISPR-SNAPf-Nup358/RanBP2 cells, the fusion protein localizes to the nuclear envelope in a punctate distribution characteristic of cytoplasmic NPC filaments. This configuration supports high-resolution fluorescence imaging, super-resolution microscopy, pulse-chase labeling, and single-molecule tracking approaches to study NPC architecture and dynamics. The flat morphology and large nuclei of U2OS cells further facilitate quantitative imaging of nuclear envelope structures.

This model enables investigation of Nup358-specific roles in CRM1/exportin-dependent nuclear export, Ran GTPase cycle regulation, and the spatial organization of cytoplasmic transport platforms. Given the involvement of Nup358 in mitotic spindle assembly and kinetochore function, the cell line is also suitable for studying cell cycle-dependent redistribution of nucleoporins and NPC disassembly/reassembly during mitosis. U2OS-CRISPR-SNAPf-Nup358/RanBP2 provides a physiologically relevant platform for dissecting structural and functional aspects of the cytoplasmic face of the nuclear pore complex in human cells.

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.

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