KC-5324

LN229-MSH2-KO Cell Line

55488

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Background of LN229-MSH2-KO Cell Line

The MSH2 (MutS homolog 2) gene plays a critical role in DNA mismatch repair (MMR), a system that corrects errors occurring during DNA replication. It encodes a protein that forms heterodimers with MSH6 or MSH3 to recognize and repair base mismatches and insertion-deletion loops. Defects in MSH2 impair MMR function, leading to microsatellite instability (MSI) and increased mutation rates, which are hallmarks of Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC). Patients with MSH2 mutations have a high lifetime risk of colorectal, endometrial, ovarian, and other cancers.

Specifications

Catalog Number	KC-5324
Cell Line Name	LN229-MSH2-KO Cell Line
Clone Number	1A1
Host Cell Line	LN229
Description	Stable LN229 cell line with MSH2 gene knockout, No.1A1
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	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+5% FBS
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:5-1:10 every 1-3 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 24 hours
Mycoplasma Status	Negative

Cell Line Generation

LN229-MSH2-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of LN229-MSH2-KO Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of LN229-MSH2-KO Cell Line stable clone using RT-PCR sequencing.

Figure 3: Characterization of LN229-MSH2-KO Cell Line stable clone using Western blot.

Cell Resuscitation

Prewarm culture medium (DMEM + 5% 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 1:5-1:10 every 1-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology. 2010 Jun;138(6):2073-2087.e3. doi: 10.1053/j.gastro.2009.12.064. PMID: 20420947; PMCID: PMC3037515.
Lynch HT, Lynch PM, Lanspa SJ, Snyder CL, Lynch JF, Boland CR. Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications. Clin Genet. 2009 Jul;76(1):1-18. doi: 10.1111/j.1399-0004.2009.01230.x. PMID: 19659756; PMCID: PMC2846640.