HK-CRISPR-mEGFP--RanBP2/Nup358 Cells

Categories: Reporter-labeled cells Description: The HK-CRISPR-mEGFP-Nup358 cell line is a genetically engineered derivative of HeLa Kyoto cells, known for their robustness and widespread use in scientific research. This cell line has been modified using CRISPR-Cas9 technology to express mEGFP (monomeric Enhanced Green Fluorescent Protein) tagged Nup358, a crucial component of the nuclear pore complex (NPC). Nup358, also known as RanBP2, plays a significant role in nucleocytoplasmic transport, mitotic spindle assembly, and other cellular processes. The mEGFP tag allows for the visualization of Nup358, facilitating real-time observation of its dynamics and interactions within the cell. HeLa Kyoto cells, a subline of the original HeLa cells, are characterized by their adaptability and stable growth in culture. The CRISPR-Cas9 system in this cell line enables precise genomic editing, ensuring the mEGFP tag is accurately fused to the Nup358 protein without disrupting its function. This makes the HK-CRISPR-mEGFP-Nup358 cell line a valuable tool for studying the structural and functional aspects of the nuclear pore complex. Researchers can use this cell line to gain insights into the mechanisms governing nucleocytoplasmic transport and the role of Nup358 in cellular homeostasis and disease states, such as cancer and viral infections. Organism: Human Tissue: Endocervix Disease: Adenocarcinoma Age: 30 years Gender: Female Ethnicity: African American Morphology: Epithelial-like cells with mosaic stone shape Growth Properties: Adherent Citation: HK-CRISPR-mEGFP-Nup358 (Cytion catalog number 301575) Biosafety Level: 1 Ncbi_ Taxid: 9606.0 Cellosaurus Accession: CVCL_B7FS Products: EGFP (Enhanced Green Fluorescent Protein) 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 Dissociation Reagent: Accutase Subculturing: Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium. 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. 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. 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