KC-3300

CHOK1-GUCY2C Cell Line

34300

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Background of CHOK1-GUCY2C Cell Line

Guanylate cyclase C (GUCY2C) is a transmembrane receptor expressed on the luminal aspect of the intestinal epithelium. The encoded protein activates the cystic fibrosis transmembrane conductance regulator. GUCY2C defends the intestinal barrier, opposing colitis and systemic genotoxicity and tumorigenesis. Furthermore, it is a novel target for immunotherapy and a diagnostic marker for primary and metastatic diseases.

Specifications

Catalog Number	KC-3300
Cell Line Name	CHOK1-GUCY2C Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous GUCY2C gene
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

CHOK1 GUCY2C Cell Line was generated using a lentiviral vector expressing the GUCY2C sequence.

Characterization

Figure 1: Characterization of endogenous human GUCY2C expression in CHOK1 using FACS.

Figure 2: Characterization of human GUCY2C overexpression in CHOK1 stable clones using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/mL Puromycin) 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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Lin JE, Snook AE, Li P, Stoecker BA, Kim GW, Magee MS, Garcia AV, Valentino MA, Hyslop T, Schulz S, Waldman SA. GUCY2C opposes systemic genotoxic tumorigenesis by regulating AKT-dependent intestinal barrier integrity. PLoS One. 2012;7(2):e31686. 2. Entezari AA, Snook AE, Waldman SA. Guanylyl cyclase 2C (GUCY2C) in gastrointestinal cancers: recent innovations and therapeutic potential. Expert Opin Ther Targets. 2021 May;25(5):335-346. 3. Cohen MB, Gold BD, Xanthakos SA, CaJacob N, Weissman T, Bartolini W, Boinpally R, Mallick M, Reasner DS, O'Dea CR, Kwak H, Ge P. Intestinal Guanylate Cyclase-C mRNA Expression in Duodenum and Colon of Children. J Pediatr Gastroenterol Nutr. 2021 Dec 1;73(6):703-709.