KC-4028

HCT116-KRAS-KO-1C3 Cell Line

40514

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Background of HCT116-KRAS-KO-1C3 Cell Line

KRAS has been identified as a KRAS-1 pseudogene on short arm of chromosome 6 and KRAS-2 gene, located on the short arm of chromosome 12 (12p11.1-12p12.1). KRAS-2 coding region spans across six exons and measures over 45 kB. The two protein isoforms of KRAS-2, KRAS-4A and KRAS-4B are produced due to alternative splicing on its fourth exon, leading to 188 and 189 monomeric amino acid sequences, respectively. For clinical and research purposes, the term KRAS refers to KRAS-4B, which constitutes the major transcriptomic product in human cells. Mutations in KRAS are among the most frequent aberrations in cancer, including colon cancer. KRAS direct targeting is daunting due to KRAS protein resistance to small molecule inhibition. Moreover, its elevated affinity to cellular guanosine triphosphate (GTP) has made the design of specific drugs challenging. Indeed, KRAS was considered 'undruggable'.Drugs targeted to block KRAS effector pathways could be combined with direct KRAS inhibitors, immunotherapy or T cell-targeting approaches in KRAS-mutant tumors. The development of valuable combination regimens will be essential against potential mechanisms of resistance that may arise during treatment.

Specifications

Catalog Number	KC-4028
Cell Line Name	HCT116-KRAS-KO-1C3 Cell Line
Host Cell Line	HCT116
Description	Stable HCT116 clone with human KRAS gene knockout, No.1C3
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	NA

Cell Line Generation

Characterization

Figure 1: Characterization of HCT116-KRAS-KO-1C3 cell line stable clone using PCR sequencing.

Figure 2: Characterization of HCT116-KRAS-KO-1C3 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of HCT116-KRAS-KO-1C3 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

Negri F, Bottarelli L, de'Angelis GL, Gnetti L. KRAS: A Druggable Target in Colon Cancer Patients. Int J Mol Sci. 2022 Apr 8;23(8):4120. doi: 10.3390/ijms23084120. PMID: 35456940; PMCID: PMC9027058.
Parikh K, Banna G, Liu SV, Friedlaender A, Desai A, Subbiah V, Addeo A. Drugging KRAS: current perspectives and state-of-art review. J Hematol Oncol. 2022 Oct 25;15(1):152. doi: 10.1186/s13045-022-01375-4. PMID: 36284306; PMCID: PMC9597994.