U2OS-CRISPR-TPR-SNAP

U2OS-CRISPR-TPR-SNAP is a genome-edited human osteosarcoma cell line derived from U2OS cells in which the endogenous TPR (Translocated Promoter Region) gene has been modified using CRISPR/Cas9 technology to encode an in-frame SNAP tag. TPR is a large coiled-coil nucleoporin that localizes to the nuclear basket on the nucleoplasmic side of the nuclear pore complex (NPC). By tagging TPR at its endogenous locus, the fusion protein is expressed under native regulatory control, preserving physiological expression levels and maintaining proper incorporation into the nuclear basket structure.

The SNAP tag enables covalent labeling of TPR with benzylguanine-conjugated fluorescent substrates in live or fixed cells, allowing highly specific and stable visualization. In U2OS-CRISPR-TPR-SNAP cells, labeled TPR displays a characteristic punctate ring-like distribution at the nuclear envelope, corresponding to NPC-associated nuclear basket structures. This system is well suited for quantitative fluorescence microscopy, super-resolution imaging, pulse-chase labeling, and dynamic studies of nuclear basket assembly and turnover. The flat morphology and large nuclei of U2OS cells facilitate high-resolution imaging of nuclear envelope-associated structures.

TPR plays critical roles in mRNA export, nuclear transport regulation, chromatin organization at the nuclear periphery, and spatial genome organization. TPR is also implicated in the formation of nuclear transport-related subcompartments and in the exclusion of heterochromatin from nuclear pore-associated regions. U2OS-CRISPR-TPR-SNAP provides a physiologically relevant model for dissecting the architecture and dynamics of the nuclear basket, investigating nucleocytoplasmic trafficking mechanisms, and studying nuclear envelope-associated chromatin interactions under endogenous expression conditions.

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.