KC-1459-DW

HCT116-MTAP-KO Cell Line

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Background of HCT116-MTAP-KO Cell Line

MTAP (methylthioadenosine phosphorylase) is encoded by the MTAP gene located on chromosome 9p21.3 and serves as a key metabolic enzyme. This chromosomal region often undergoes deletion in various cancers, including glioblastoma, mesothelioma, and non-small cell lung cancer, frequently co-occurring with CDKN2A/B gene deletions. The MTAP enzyme catalyzes the phosphorylation of 5\'-deoxy-5\'-methylthioadenosine (MTA), a byproduct of polyamine synthesis, to yield adenine and methylthioribose-1-phosphate. This reaction is essential for recycling purine precursors and maintaining cellular methionine levels, linking MTAP activity to nucleotide biosynthesis and epigenetic regulation mediated by S-adenosylmethionine (SAM).

Specifications

Catalog Number	KC-1459-DW
Cell Line Name	HCT116-MTAP-KO Cell Line
Clone Number	3B4
Host Cell Line	HCT116
Description	Stable HCT116 clone with human MTAP gene knockout, No.3B4
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	McCoy's 5A+20% FBS+10% DMSO
Propagation Medium	McCoy's 5A+10% FBS
Selection Marker	NA
Morphology	Fibroblast
Subculture	Split saturated culture 1:4-1:5 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	Yes

Cell Line Generation

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

Characterization

Figure 1: Characterization of HCT116-MTAP-KO-3B4 cell line stable clone using PCR sequencing.

Figure 2: Characterization of HCT116-MTAP-KO-3B4 cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (McCoy's 5A + 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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% McCoy's 5A + 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

Miao D, Li Y, Shen X, et al. MTAP deletion and immune checkpoint inhibitor resistance in solid tumors. J Clin Oncol. 2020 Mar 15;38(15_suppl):11546. doi:10.1200/JCO.2020.38.15_suppl.11546. PMID:32256891.
Vazquez F, Zhang R, Qin X, et al. Targeting MTAP-deficient tumors through metabolic rewiring. Cancer Discov. 2021 Aug;11(8):1972-1990. doi:10.1158/2159-8290.CD-20-1582. Epub 2021 Jun 14. PMID:34136785; PMCID:PMC8363878.