NRK-4xlambdaN22-3xmEGFP-M9 Cells

Categories: Reporter-labeled cells Description: 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 4xlambdaN22-3xmEGFP-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. However, it is important to note that this cell line exhibits some variegation, which refers to the variability in gene expression levels among individual cells within the population. 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. Organism: Rat Tissue: Kidney Synonyms: NRK 4xlN22-3xmEGFP-M9 Breed: OsborneMendel Morphology: Fibroblast-like cells with fusiform shape Growth Properties: Monolayer, adherent Citation: NRK-4xlambdaN22-3xmEGFP-M9 (Cytion catalog number 500672) Biosafety Level: 1 Ncbi_ Taxid: 10116.0 Cellosaurus Accession: CVCL_AV97 Receptors Expressed: Epidermal growth factor (EGF), multiplication stimulating activity (MSA) Protein Expression: 4xlambdaN22-3xmEGFP-M9: Location/Gene: 937..1009, 1066..1138, 1194..1261, 1323..1390 / lambda peptide, 1462..2176, 2179..2890, 2896..3612 / mEGFP, 3612..3815 / M9-His, 5090..5884 / KanR/NeoR, 7195..584 / Pcmv Products: M9-His tag between BsrG1/HindIII, Neomycin, Phosphotransferase, CMV Promotor Culture Medium: DMEM, w: 4.5 g/L Glucose, w: 4 mM L-Glutamine, w: 3.7 g/L NaHCO3, w: 1.0 mM Sodium pyruvate (Cytion article number 820300a) Supplements: Supplement the medium with 10% FBS, 0.5 mg/mL G418 Dissociation Reagent: Accutase Subculturing: Discard the old medium and wash the cells with PBS. Add a freshly prepared 0.025% trypsin/0.02% EDTA solution heated to 37 degrees Celsius and wait until the cells detach, which usually takes about 5 minutes. Neutralize the trypsin by adding fresh medium, then transfer the cell mixture to a tube and centrifuge. After centrifugation, remove the supernatant, resuspend the cell pellet in fresh culture medium, and transfer the suspension to new flasks. Incorporate G418 into the culture medium to achieve a final concentration of 0.5 mg/ml Seeding Density: 2 to 4 x 104 cells/cm2 Fluid Renewal: 2 to 3 times per week Freeze Medium: 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. Thawing And Culturing Cells: Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit. 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. 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. Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening. 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. Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium. 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. Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes. Sterility: 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. Safety Precautions: When planning to store a cryovial in liquid nitrogen for future thawing, it is mandatory to adhere to stringent safety measures. Appropriate protective gloves and clothing are essential, and the use of a face mask or safety goggles is required during the transfer of frozen samples to or from the liquid nitrogen tank. This is to mitigate the risk of injury from potential cryovial explosions upon removal, which can result in the projection of sharp fragments. Disclaimer: Our cells are provided for in vitro laboratory research purposes exclusively and are not intended for clinical or diagnostic use, nor are they to be administered to humans or used for veterinary purposes. Users must adhere to all applicable guidelines and regulations for the handling and use of these cells in a research setting. Warranty: We stand by the promise of delivering products with high cell viability and robust culture performance. To achieve the best results, please make sure you follow the storage and culture instructions detailed in the product information sheet closely. Your adherence to these guidelines is key to success. Certificate Of Analysis: The certificate of analysis can be requested on the website or via email at info@cytion.com. Please indicate the lot number of your product in the email. Subject To Third- Party Agreements: Please note that this cell line is not available under a standard Cytion MTA, as it requires a third-party agreement and/or is subject to negotiation with the original licensor. Required Product 1: 820300a Required Product 3: 860015.0 Required Product 4: 830100.0