KC-6116

HCT116-WRN-G729S-KI Cell Line

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Background of HCT116-WRN-G729S-KI Cell Line

Located on human chromosome 8p12, the WRN gene belongs to the RecQ helicase family, encoding a protein with helicase and exonuclease activities critical for DNA repair, telomere maintenance, and genome stability. Biallelic WRN mutations cause Werner syndrome, an adult-onset premature aging disorder with symptoms like premature graying and cataracts. The rare homozygous G729S missense mutation links to a distinct phenotype: progressive pulmonary failure, recurrent spontaneous pneumothorax, and interstitial lung disease (ILD) in adolescents/young adults. Unlike classic Werner syndrome, it lacks typical premature aging features. In silico analyses suggest G729S impairs WRN’s exonuclease activity or stability, disrupting DNA repair/telomere pathways, highlighting WRN’s pleiotropic roles.

Specifications

Catalog Number	KC-6116
Cell Line Name	HCT116-WRN-G729S-KI Cell Line
NCBI/UniProt Accession Number	7486
Clone Number	1A2
Host Cell Line	HCT116
Description	Stable HCT116 clone expressing endogenous WRN gene bearing G729S mutations, No.1A2
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	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 27 hours
Mycoplasma Status	Negative

Cell Line Generation

HCT116-WRN-G729S-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of HCT116-WRN-G729S-KI Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of HCT116-WRN-G729S-KI Cell Line stable clone using RT PCR sequencing.

Figure 3: Characterization of Dose-response curves and IC50 values for HCT116 and HCT116-WRN-G729S-KI cells treated with VVD-214 and HRO761 over 8 days.

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 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (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

Sezer A, Kayhan G, Gursoy TR, Eyuboglu TS, Percin FE. A homozygous missense variant in the WRN gene segregating in a family with progressive pulmonary failure with recurrent spontaneous pneumothorax and interstitial lung disease. Am J Med Genet A. 2023 Jan;191(1):220-227. doi: 10.1002/ajmg.a.62986. Epub 2022 Oct 10. PMID: 36214313.