KC-3852

CHOK1-CRE-Luc2-cyno-GLP1R Cell Line

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Home » CHOK1-CRE-Luc2-cyno-GLP1R Cell Line

Background of CHOK1-CRE-Luc2-cyno-GLP1R Cell Line

Cyclic adenosine monophosphate (cAMP) is a second messenger involved in cell signaling that regulates variousl physiological and pathological processes. cAMP regulates the transcription of target genes by activating proteinl kinase A (PKA) and the transcription factor cAMP response element-binding protein (CREB). CRE is the target of many extracellular and intracellular signaling pathways, including cAMP, calcium,l GPCR (G-protein coupled receptors), and neurotrophins. The CAMP/PKA/CREB signaling pathway has both tumor-suppressive and tumor-promoting effects in cancer cells and can be useful in studying cancer signaling pathways. GLP1R (Glucagon-Like Peptide-1 Receptor) is a G protein-coupled receptor that plays a crucial role in glucose homeostasis and insulin secretion. It is primarily expressed in pancreatic beta cells, where it responds to the binding of glucagon-like peptide-1 (GLP-1), a hormone released from the intestine after meals. Activation of GLP1R stimulates insulin release, enhances beta cell proliferation, and suppresses glucagon secretion, thereby regulating blood glucose levels. GLP1R is also expressed in other tissues, including the brain, heart, and gastrointestinal tract, where it modulates various physiological processes. Mutations in the GLP1R gene have been linked to impaired glucose tolerance and type 2 diabetes mellitus (T2DM). Additionally, GLP1R agonists, such as exenatide and liraglutide, are widely used in the treatment of T2DM due to their ability to improve glycemic control and promote weight loss. Recent research has also explored the potential of GLP1R agonists in treating cardiovascular diseases and neurodegenerative disorders.

Specifications

Catalog NumberKC-3852
Cell Line NameCHOK1-CRE-Luc2-cyno-GLP1R Cell Line
Host Cell LineCHOK1-CRE-Luc2
DescriptionStable CHOK1-CRE-Luc2 cell line expressing exogenous cyno GLP1R gene
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% 1640+20% FBS+10% DMSO
Propagation Medium1640+10% FBS +750μg/mL Hygromycin+10μg/mL Puromycin
Selection MarkerPuromycin, Hygromycin B
MorphologyEpithelial
SubcultureSplit saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative
In Vivo ValidationNA

Cell Line Generation

CHOK1-CRE-Luc2-cyno-GLP1R cell line was generated using lentivirus expressing cyno GLP1R sequence.

Characterization

Figure: CHOK1-CRE-Luc2-cyno-GLP1R cell line was seeded into the 96-well plate, and treated with GLP1 at a maximum concentration of 100ng/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

  1. Prewarm culture medium (1640 supplemented with 10% FBS, 750μg/mL Hygromycin 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% 1640 + 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. Ast J, Broichhagen J, Hodson DJ. Reagents and models for detecting endogenous GLP1R and GIPR. EBioMedicine. 2021 Dec;74:103739. doi: 10.1016/j.ebiom.2021.103739. Epub 2021 Dec 12. PMID: 34911028; PMCID: PMC8669301.
  2. Campbell JE, Müller TD, Finan B, DiMarchi RD, Tschöp MH, D'Alessio DA. GIPR/GLP-1R dual agonist therapies for diabetes and weight loss-chemistry, physiology, and clinical applications. Cell Metab. 2023 Sep 5;35(9):1519-1529. doi: 10.1016/j.cmet.2023.07.010. Epub 2023 Aug 16. PMID: 37591245; PMCID: PMC10528201.
  3. Zhang H, Kong Q, Wang J, Jiang Y, Hua H. Complex roles of cAMP-PKA-CREB signaling in cancer. Exp Hematol Oncol. 2020 Nov 24;9(1):32. doi: 10.1186/s40164-020-00191-1. PMID: 33292604; PMCID: PMC7684908.
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