KC-3435

293T-CCKBR Cell Line

34388

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Background of 293T-CCKBR Cell Line

The CCKBR gene encodes a G protein-coupled receptor that functions as a B-type gastrin receptor, exhibiting high affinity for both sulfated and non-sulfated cholecystokinin (CCK) analogs. This receptor is predominantly expressed in the central nervous system and the gastrointestinal tract. CCKBR has been implicated in diseases such as cocaine addiction and obsessive-compulsive disorder in humans. Additionally, it plays a role in gastric cancer, where its overexpression may contribute to tumor growth. Currently, there are studies focusing on developing antagonists like L-365260, which shows potential as an orally effective and selective non-peptide antagonist for CCK-B receptors, aiming at therapeutic targets for conditions associated with CCKBR overactivity.

Specifications

Catalog Number	KC-3435
Cell Line Name	293T-CCKBR Cell Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human CCKBR 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/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

293T-CCKBR cell line was generated using a lentiviral vector expressing the human CCKBR sequence.

Characterization

Figure 1: Characterization of CCKBR overexpression in the 293T-CCKBR stable clone using qPCR.

Figure 2: Characterization of CCKBR overexpression in the 293T-CCKBR stable clone using WB.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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% 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. Liu C, Chen K, Wang H, Zhang Y, Duan X, Xue Y, He H, Huang Y, Chen Z, Ren H, Wang H, Zeng C. Gastrin Attenuates Renal Ischemia/Reperfusion Injury by a PI3K/Akt/Bad-Mediated Anti-apoptosis Signaling. Front Pharmacol. 2020 Nov 6;11:540479. doi: 10.3389/fphar.2020.540479. PMID: 33343341; PMCID: PMC7740972. 2. Jose PA, Yang Z, Zeng C, Felder RA. The importance of the gastrorenal axis in the control of body sodium homeostasis. Exp Physiol. 2016 Apr;101(4):465-70. doi: 10.1113/EP085286. Epub 2016 Mar 2. PMID: 26854262; PMCID: PMC4818653.