NRK-4xlambdaN22-3xmEGFP-M9

The NRK-4xlambdaN22-3xmEGFP-M9 cell line is a clonal stable cell line derived from normal rat kidney (NRK) cells through the transfection of a circular plasmid. This plasmid contains genetic constructs encoding four tandem repeats of lambda N22 RNA-binding sites and three tandem repeats of mEGFP (monomeric enhanced green fluorescent protein) tags fused with the M9 nuclear localization signal. Post-transfection, the cells underwent drug resistance selection to ensure the stability of the genetic modifications.

Approximately 50% of the cells in this clonal stable line express the fluorescent marker 4xλN22-3xmEGFP-M9, indicating successful incorporation of the plasmid. The expression of this marker allows for real-time visualization of intracellular processes, facilitated by the robust fluorescent signal of mEGFP. The M9 nuclear localization signal ensures that the expressed fusion proteins are transported to the nucleus, making this cell line particularly useful for studying nuclear-cytoplasmic transport, RNA dynamics, and gene expression regulation.

This NRK-4xlambdaN22-3xmEGFP-M9 cell line is valuable for researchers focusing on RNA-binding protein interactions, RNA metabolism, and the mechanisms underlying nuclear import and export. The presence of the mEGFP marker enables advanced imaging techniques such as confocal microscopy and live-cell imaging, providing detailed insights into the spatial and temporal dynamics of cellular components. Despite the variegation, the cell line remains a powerful tool for dissecting complex molecular pathways and understanding cellular functions at a deeper level.

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.