KC-4044

293T-TP53-Y220C-Cell-Line

40546

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Background of 293T-TP53-Y220C-Cell-Line

The TP53 gene, also known as P53, is an oncogene that encodes a protein with a molecular weight of 53 kDa. Mutations in this gene occur in more than 50% of all malignant tumors. The protein encoded by this gene is a transcription factor that controls the initiation of the cell cycle, and plays a decisive role from the beginning of cell division. If the cell is damaged and cannot be repaired, the P53 protein is involved in the initiation process, causing the cell to die in apoptosis.

Specifications

Catalog Number	KC-4044
Cell Line Name	293T-TP53-Y220C-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous TP53 Y220C gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-TP53-Y220C cell line was generated using a lentiviral vector expressing the TP53 Y220C sequence.

Characterization

Figure 1: Characterization of TP53 overexpression in the 293T TP53 Y220C stable clone using qPCR

Figure 2: Characterization of TP53 Y220C in the 293T TP53 Y220C stable clone using PCR sequence.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Voskarides K, Giannopoulou N. The Role of TP53 in Adaptation and Evolution. Cells. 2023 Feb 3;12(3):512. doi: 10.3390/cells12030512. PMID: 36766853; PMCID: PMC9914165.
Testa U, Castelli G, Pelosi E. TP53-Mutated Myelodysplasia and Acute Myeloid Leukemia. Mediterr J Hematol Infect Dis. 2023 Jul 1;15(1):e2023038. doi: 10.4084/MJHID.2023.038. PMID: 37435040; PMCID: PMC10332352.
Hunter AM, Sallman DA. Targeting TP53 Mutations in Myelodysplastic Syndromes. Hematol Oncol Clin North Am. 2020 Apr;34(2):421-440. doi: 10.1016/j.hoc.2019.11.004. Epub 2019 Dec 12. PMID: 32089220.