KC-5600

HCT116-WRN-G729D-KI Cell Line

58220

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Background of HCT116-WRN-G729D-KI Cell Line

The Werner syndrome ATP-dependent helicase (WRN) gene encodes a RecQ family DNA helicase-exonuclease critical for maintaining genomic stability. WRN participates in DNA repair, replication, recombination, and telomere maintenance, with its loss-of-function mutations causing Werner syndrome—a premature aging disorder characterized by genomic instability and increased cancer susceptibility. WRN-deficient cells exhibit hypersensitivity to DNA-damaging agents and impaired resolution of replication stress, highlighting its essential role in DNA damage response. Recent studies reveal synthetic lethality between WRN deficiency and microsatellite instability (MSI) in cancers, suggesting WRN as a promising therapeutic target for MSI-high tumors. Small-molecule WRN inhibitors are under investigation for selective anticancer effects. Further research is needed to elucidate WRN's multifaceted roles in aging and cancer, and to develop targeted therapies leveraging WRN dependency in specific cancer contexts.

Specifications

Catalog Number	KC-5600
Cell Line Name	HCT116-WRN-G729D-KI Cell Line
Clone Number	1C6
Host Cell Line	HCT116
Description	Stable HCT116 clone expressing endogenous WRN gene bearing G729D mutations, No.1C6
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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

HCT116-WRN-G729D-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of HCT116-WRN-G729D-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of HCT116-WRN-G729D-KI cell line stable clone using RT-PCR sequencing.

Figure 3. Characterization of dose-response curves for KRAS inhibitors on HCT116 and HCT116-WRN-G729D-KI cells.

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 1:3-1:6 every 2-3 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

Oshima, J., et al. (2017). "Werner syndrome: clinical features, pathogenesis and potential therapeutic interventions." Ageing Research Reviews, 33, 105-114.
van Wietmarschen, N., et al. (2020). "BLM and WRN DNA helicases mitigate R-loops to maintain genome stability." Nature Communications, 11(1), 4281.
Lieb, S., et al. (2023). "Werner syndrome helicase is a synthetic lethal target in cancers with microsatellite instability." Nature, 614(7948), 562-570.