KC-4384

T47D-CCNE1-Flag Cell Line

60716

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Background of T47D-CCNE1-Flag Cell Line

CCNE1 (Cyclin E1) is a Protein Coding gene. It also known as CCNE, the protein encoded by this gene belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Cyclin E, while currenly not as widely implicated as its cyclin D counterparts, has been implicated in various carcinomas, including breast, gastric, stomach and colorectal. High levels of cyclin E, either by gene amplification or overexpression, have been shown to lead to poorer prognosis in gastic carcinoma, and these measurements are correlated with later stage disease. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. This cyclin forms a complex with and functions as a regulatory subunit of CDK2, whose activity is required for cell cycle G1/S transition. Diseases associated with CCNE1 include Hereditary Breast Ovarian Cancer Syndrome and Pancreatic Cancer.

Specifications

Catalog Number	KC-4384
Cell Line Name	T47D-CCNE1-Flag Cell Line
NCBI/UniProt Accession Number	NM_001238
Clone Number	7#
Host Cell Line	Human T47D Cell Line
Description	Stable T47D clone expressing exogenous CCNE1-Flag 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 1μg/mL Puromycin +0.2Units/mL Insulin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:3 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

Cell Line Generation

T47D-CCNE1-Flag cell line was generated using a lentiviral vector expressing the CCNE1-Flag sequence.

Characterization

Figure 1: T47D cells expressing CCNE1 Flag were seeded into 96-well plates, treated with compounds for 240 hours, and then read out with Cell-Titer Glo system.

Figure 2: Characterization of CCNE1-Flag overexpression in the T47D-CCNE1-Flag stable clone using Western Blot.

Figure 3: Characterization of CCNE1-Flag in the T47D stable clone using PCR sequencing.

Figure 4: Tumor growth and body weight curves of B-NDG nude mice inoculated with T47D-CCNE1-Flag cells (n=5).

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 1μg/mL Puromycin +0.2Units/mL Insulin )in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:2-1:3 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Gallo D, Young JTF, Fourtounis J, Martino G, Álvarez-Quilón A, Bernier C, Duffy NM, Papp R, Roulston A, Stocco R, Szychowski J, Veloso A, Alam H, Baruah PS, Fortin AB, Bowlan J, Chaudhary N, Desjardins J, Dietrich E, Fournier S, Fugère-Desjardins C, Goullet de Rugy T, Leclaire ME, Liu B, Bhaskaran V, Mamane Y, Melo H, Nicolas O, Singhania A, Szilard RK, Tkáč J, Yin SY, Morris SJ, Zinda M, Marshall CG, Durocher D. CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition. Nature. 2022 Apr;604(7907):749-756. doi: 10.1038/s41586-022-04638-9. Epub 2022 Apr 20. PMID: 35444283; PMCID: PMC9046089..
2.Zheng X, Chen L, Liu W, Zhao S, Yan Y, Zhao J, Tian W, Wang Y. CCNE1 is a predictive and immunotherapeutic indicator in various cancers including UCEC: a pan-cancer analysis. Hereditas. 2023 Mar 24;160(1):13. doi: 10.1186/s41065-023-00273-0. PMID: 36964635; PMCID: PMC10037856.
3.House NC, Brown VE, Chen M, Yuan L, Moore SL, Guo J, Choi YJ, Muthuswamy L, Ribich S, Ramsden P, Faia KL. Profiling the Activity of the Potent and Highly Selective CDK2 Inhibitor BLU-222 Reveals Determinants of Response in CCNE1-Aberrant Ovarian and Endometrial Tumors. Cancer Res. 2025 Apr 3;85(7):1297-1309. doi: 10.1158/0008-5472.CAN-24-2360. PMID: 39945650; PMCID: PMC11967718.