KC-5927

HCT116-BAP1-KO Cell Line

59374

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Background of HCT116-BAP1-KO Cell Line

The BRCA1 Associated Protein 1 (BAP1) gene (located on chromosome 3p21.1) encodes a nuclear deubiquitinating enzyme (DUB) functioning as a potent tumor suppressor. As part of the Polycomb repressive deubiquitination complex (PR‑DUB), BAP1 regulates key cellular processes including cell cycle progression, DNA damage repair, ferroptosis, and chromatin modulation. Inactivating germline or somatic mutations in BAP1 significantly predispose individuals to various cancers, most notably malignant mesothelioma, uveal melanoma, clear cell renal cell carcinoma, and specific types of cutaneous melanoma (BAP1‑inactivated melanocytic tumors). BAP1 mutation status is increasingly recognized as a critical factor for cancer risk assessment, prognosis, and potential therapeutic targeting.

Specifications

Catalog Number	KC-5927
Cell Line Name	HCT116-BAP1-KO Cell Line
Clone Number	1C1
Host Cell Line	HCT116
Description	Stable HCT116 cell line with BAP1 gene knockout, No.1C1
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
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:5-1:10 every 1-2 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 27 hours
Mycoplasma Status	Negative

Cell Line Generation

HCT116-BAP1-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of HCT116-BAP1-KO Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of HCT116-BAP1-KO Cell Line stable clone using RT-PCR sequencing.

Figure 3: Characterization of HCT116-BAP1-KO Cell Line stable clone using Western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS) 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 1:5-1:10 every 1-2 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Carbone M, Yang H, Pass HI, Krausz T, Testa JR, Gaudino G. BAP1 and cancer. Nat Rev Cancer. 2013 Mar;13(3):153-9. doi: 10.1038/nrc3459. PMID: 23550303; PMCID: PMC3792854.
Zhang Y, Shi J, Liu X, Feng L, Gong Z, Koppula P, Sirohi K, Li X, Wei Y, Lee H, Zhuang L, Chen G, Xiao ZD, Hung MC, Chen J, Huang P, Li W, Gan B. BAP1 links metabolic regulation of ferroptosis to tumour suppression. Nat Cell Biol. 2018 Oct;20(10):1181-1192. doi: 10.1038/s41556-018-0178-0. Epub 2018 Sep 10. PMID: 30202049; PMCID: PMC6170713.
Walpole S, Pritchard AL, Cebulla CM, Pilarski R, Stautberg M, Davidorf FH, de la Fouchardière A, Cabaret O, Golmard L, Stoppa-Lyonnet D, Garfield E, Njauw CN, Cheung M, Turunen JA, Repo P, Järvinen RS, van Doorn R, Jager MJ, Luyten GPM, Marinkovic M, Chau C, Potrony M, Höiom V, Helgadottir H, Pastorino L, Bruno W, Andreotti V, Dalmasso B, Ciccarese G, Queirolo P, Mastracci L, Wadt K, Kiilgaard JF, Speicher MR, van Poppelen N, Kilic E, Al-Jamal RT, Dianzani I, Betti M, Bergmann C, Santagata S, Dahiya S, Taibjee S, Burke J, Poplawski N, O'Shea SJ, Newton-Bishop J, Adlard J, Adams DJ, Lane AM, Kim I, Klebe S, Racher H, Harbour JW, Nickerson ML, Murali R, Palmer JM, Howlie M, Symmons J, Hamilton H, Warrier S, Glasson W, Johansson P, Robles-Espinoza CD, Ossio R, de Klein A, Puig S, Ghiorzo P, Nielsen M, Kivelä TT, Tsao H, Testa JR, Gerami P, Stern MH, Paillerets BB, Abdel-Rahman MH, Hayward NK. Comprehensive Study of the Clinical Phenotype of Germline BAP1 Variant-Carrying Families Worldwide. J Natl Cancer Inst. 2018 Dec 1;110(12):1328-1341. doi: 10.1093/jnci/djy171. PMID: 30517737; PMCID: PMC6292796.
Louie BH, Kurzrock R. BAP1: Not just a BRCA1-associated protein. Cancer Treat Rev. 2020 Nov;90:102091. doi: 10.1016/j.ctrv.2020.102091. Epub 2020 Aug 20. PMID: 32877777; PMCID: PMC7655689.