KC-5269

HuH7-CLDN6-KO Cell Line

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Background of HuH7-CLDN6-KO Cell Line

The CLDN6 gene encodes claudin-6, a member of the claudin family of tight junction proteins, which play critical roles in maintaining cell polarity and barrier function in epithelial tissues. Claudin-6 is primarily expressed during embryonic development and is often silenced in adult tissues, but its re-expression has been observed in various cancers, including gastric, ovarian, and testicular cancers, suggesting a potential role in tumorigenesis.

Specifications

Catalog Number	KC-5269
Cell Line Name	HuH7-CLDN6-KO Cell Line
Clone Number	1A4
Host Cell Line	HuH7
Description	Stable HuH7 cell line with CLDN6 gene knockout, No.1A4
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+10% 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 22 hours
Mycoplasma Status	Negative

Cell Line Generation

HuH7-CLDN6-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of HuH7-CLDN6-KO Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of HuH7-CLDN6-KO Cell Line stable clone using RT-PCR sequencing.

Figure 3: Characterization of HuH7-CLDN6-KO Cell Line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 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 1:5-1:10 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

https://www.ncbi.nlm.nih.gov/gene/9074
Turksen K, Troy TC. Claudin-6: a novel tight junction molecule is developmentally regulated in mouse embryonic epithelium. Dev Dyn. 2001 Oct;222(2):292-300. doi: 10.1002/dvdy.1174. PMID: 11668606.