KC-3195

CHOK1-CRE-Luc2-GCGR Cell Line

34208

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Background of CHOK1-CRE-Luc2-GCGR Cell Line

Cyclic adenosine monophosphate (cAMP), the first discovered second messenger, plays a key role in cell signaling, regulating many physiological and pathological processes. cAMP can regulate the transcription of a variety of target genes, primarily through protein kinase A (PKA) and its downstream effectors such as cAMP-responsive element binding protein (CREB). Abnormal elevations of glucagon (GCG) are the leading cause of type II diabetes. When GCG interacts with glucagon receptor (GCGR), GCG can increase blood sugar levels.

Specifications

Catalog Number	KC-3195
Cell Line Name	CHOK1-CRE-Luc2-GCGR Cell Line
Clone Number	2#
Host Cell Line	CHOK1-CRE-Luc2
Description	Stable CHOK1-CRE-Luc2 cell line expressing exogenous GCGR 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+750µg/mL Hygromycin B
Selection Marker	Puromycin, Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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-CRE-Luc2-GCGR Cell Line was generated using a lentiviral vector expressing GCGR sequence.

Characterization

Figure 1. Characterization of human GCGR overexpression in the CHOK1-CRE-Luc2 stable clone using FACS.

Figure 2. CHOK1-CRE-Luc2-GCGR cells were seeded into the 96-well plate, and treated with GC 6 hours, then readout with Bright-lite™ Luciferase Assay system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10%FBS, 750µg/mL Hygromycin B, 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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (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.Sands WA, Palmer TM. Regulating gene transcription in response to cyclic AMP elevation. Cell Signal. 2008 Mar;20(3):460-6. doi: 10.1016/j.cellsig.2007.10.005. Epub 2007 Oct 12. PMID: 17993258. 2.Verleye L, Ottevanger PB, van der Graaf W, Reed NS, Vergote I; Gynaecological Cancer Group (GCG) of European Organisation for Research and Treatment of Cancer (EORTC). EORTC-GCG process quality indicators for ovarian cancer surgery. Eur J Cancer. 2009 Mar;45(4):517-26. doi: 10.1016/j.ejca.2008.09.031. Epub 2008 Nov 14. PMID: 19013789.