KC-4274

293T-WRN-C727A-KI-3B1 Cell Line

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Background of 293T-WRN-C727A-KI-3B1 Cell Line

The WRN gene, encoding the Werner syndrome RecQ-like helicase (WRN), is a member of the RecQ helicase family. This protein plays a crucial role in maintaining genomic stability through its involvement in DNA repair, replication, and telomere maintenance. Mutations in the WRN gene are responsible for Werner syndrome, a rare autosomal recessive disorder characterized by premature aging and an increased risk of cancer. Individuals with Werner syndrome exhibit symptoms such as early onset of atherosclerosis, type 2 diabetes, osteoporosis, and cataracts, typically developing these conditions in their 20s or 30s.

Specifications

Catalog Number	KC-4274
Cell Line Name	293T-WRN-C727A-KI-3B1 Cell Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous WRN gene bearing C727A mutations, No.3B1
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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	N/A
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
In Vivo Validation	NA

Cell Line Generation

293T-WRN-C727A-KI-3B1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-WRN-C727A-KI-3B1 cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-WRN-C727A-KI-3B1 cell line stable clone using RT-PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Opresko, P. L., et al. (2014). The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2.Molecular Cell, 54(6), 1163-1174. https://doi.org/10.1016/j.molcel.2014.05.018
2. Monnat, R. J., Jr. (2010). Human RECQ helicases: roles and mechanisms.Nature Reviews Molecular Cell Biology, 11(10), 721-733. https://doi.org/10.1038/nrm2970
3.Oshima, J., et al. (2017). Werner syndrome: a review on clinical features, molecular mechanisms, and potential therapies.Aging and Disease, 8(5), 624-644. https://doi.org/10.14336/AD.2017.0115