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KC-3323

CHOK1-CRE-Luc2-PTH1R-High Cell Line

Home » CHOK1-CRE-Luc2-PTH1R-High Cell Line

Background

Cyclic adenosine monophosphate (cAMP) plays a key role in signal transduction pathways as a second messenger. cAMP can regulate the transcription of various target genes, mainly through protein kinase A (PKA) and its downstream effectors such as cAMP-responsive element binding protein (CREB). In addition, PKA can phosphorylate many kinases such as Raf, GSK4 and FAK. Aberrant cAMP-PKA signaling is involved in various types of diseases, such as prefrontal cortex disorders, chronic inflammatory diseases, chronic lymphocytic leukemia (CLL) and human tumors. Especially, cAMP signaling may have both tumor-suppressive and tumor-promoting roles depending on the tumor types and context. cAMP-PKA signaling can regulate cancer cell growth, migration, invasion and metabolism. PTH1R, also known as PTHR1, is a protein that belongs to the G-protein coupled receptor 2 family. PTH1R is a receptor for parathyroid hormone and for parathyroid hormone-related peptide. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase, and phosphatidylinositol-calcium second messenger system. Diseases associated with PTH1R include Jansen type metaphyseal chondrodysplasia and Blomstrand type Chondrodysplasia. Among its related pathways are endochondral ossification with skeletal dysplasias and GPCR downstream signaling.

Specifications

Catalog Number:
KC-3323
Cell Line Name:
CHOK1-CRE-Luc2-PTH1R-High Cell Line
Host Cell Line:
Description:
Stable CHO-K1 cell line expressing exogenous luciferase under the control of cAMP response element (CRE) and exogenous human PTH1R gene
Quantity:
One vial of frozen cells (5×106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% RPMI1640+20% FBS+10% DMSO
Propagation Medium:
RPMI1640+10%FBS+10µg/mL Puromycin+750µg/mL Hygromycin B
Selection Marker:
Puromycin, HygromycinB
Morphology:
Epithelial-like
Subculture:
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 105 cells/mL
Incubation:
37 °C with 5% CO2
Storage:
Liquid nitrogen immediately upon receiving
Doubling Time:
Approximately 24 hours
Mycoplasma Status:
Negative

Cell Line Generation

CHOK1-CRE-Luc2-PTH1R reporter cell line was generated using a lentiviral method.

Characterization

Application

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 10µg/mL Puromycin + 750µg/mL Hygromycin B) 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 1-2 × 105 cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI-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. Kim N, Shin S, Bae SW. cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins. Biosensors (Basel), 2021.
2. Zhang H, Kong Q, Wang J, Jiang Y, Hua H. Complex roles of cAMP-PKA-CREB signaling in cancer. Exp Hematol Oncol, 2020.
3. Pacini ESA, Satori NA, Jackson EK, Godinho RO. Extracellular cAMP-Adenosine Pathway Signaling: A Potential Therapeutic Target in Chronic Inflammatory Airway Diseases, 2022.
4. Murray F, Insel PA. Targeting cAMP in chronic lymphocytic leukemia: a pathway-dependent approach for the treatment of leukemia and lymphoma. Expert Opin Ther Targets, 2014.
5. Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2. PMID: 18611381 PMCID: PMC2601695 DOI: 10.1016/j.str.2008.04.010.
6. Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation. PMID: 20172855 PMCID: PMC2852981 DOI: 10.1074/jbc.M109.093138.
7. Entrez Gene: PTH1R parathyroid hormone 1 receptor.
8. Yamaguchi T, Hosomichi K, Narita A, Shirota T, Tomoyasu Y, Maki K, Inoue I (Jul 2011). Exome resequencing combined with linkage analysis identifies novel PTH1R variants in primary failure of tooth eruption in Japanese. Journal of Bone and Mineral Research. 26 (7): 1655ÿ61. doi:10.1002/jbmr.385. PMID 21404329. S2CID 23855913.
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