KC-3700

293T CRE Luc2 RAMP3 CALCRL Cell Line

34626

Home » 293T CRE Luc2 RAMP3 CALCRL Cell Line

Background of 293T CRE Luc2 RAMP3 CALCRL 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. CALCRL encodes a G-protein-coupled seven-transmembrane domain receptor, which requires one of three single transmembrane domain co-receptors, RAMP1, RAMP2, or RAMP3, for cell surface expression and binding of its peptide ligands. RAMP3 is a member of the receptor activity modifying protein (RAMPs) family. RAMP3 with the calcitonin receptor, produces an amylin receptor complex (AMY3). Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. Pramlintide, a nonamyloidogenic analogue of human amylin is approved for the treatment of type 1 diabetes in combination with insulin. Adrenomedullin (ADM) binds to CALCRL/ RAMP3 complexes.

Specifications

Catalog Number	KC-3700
Cell Line Name	293T CRE Luc2 RAMP3 CALCRL Cell Line
Host Cell Line	293T-CRE-Luc2
Description	Stable 293T-CRE-Luc2 cell line expressing exogenous human RAMP3 and CALCRL gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Cell model for monitoring RAMP3 and CALCRL signaling pathway.
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +150μg/mL Hygromycin+1μg/mL Puromycin
Selection Marker	Hygromycin and 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-CRE-Luc2-RAMP3-CALCRL reporter cell line was generated using a lentiviral vector expressing the human RAMP3 and CALCRL sequence.

Characterization

Figure 1. 293T-CRE-Luc2-RAMP3-CALCRL cell line was seeded into the 96-well plate, and treated with Adrenomedullin at a maximum concentration of 10 μg/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/mL Hygromycin and 1μg/ml Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Mackie DI, Nielsen NR, Harris M, Singh S, Davis RB, Dy D, Ladds G, Caron KM. RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis. Proc Natl Acad Sci U S A. 2019 Nov 26;116(48):24093-24099. doi: 10.1073/pnas.1905561116. Epub 2019 Nov 11. PMID: 31712427; PMCID: PMC6883789.
Prakash J, Herlin M, Kumar J, Garg G, Akesson KE, Grabowski PS, Skerry TM, Richards GO, McGuigan FEA. Analysis of RAMP3 gene polymorphism with body composition and bone density in young and elderly women. Gene. 2019;721S:100009. doi: 10.1016/j.gene.2019.100009. Epub 2019 Feb 14. PMID: 34530989.
Grandits AM, Wieser R. Gene expression changes contribute to stemness and therapy resistance of relapsed acute myeloid leukemia: roles of SOCS2, CALCRL, MTSS1, and KDM6A. Exp Hematol. 2021 Jul;99:1-11. doi: 10.1016/j.exphem.2021.05.004. Epub 2021 May 21. PMID: 34029637; PMCID: PMC7612147.
Pham V, Zhu Y, Dal Maso E, Reynolds CA, Deganutti G, Atanasio S, Hick CA, Yang D, Christopoulos A, Hay DL, Furness SGB, Wang MW, Wootten D, Sexton PM. Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor. ACS Pharmacol Transl Sci. 2019 Mar 18;2(3):183-197. doi: 10.1021/acsptsci.9b00010. PMID: 32219220; PMCID: PMC7088965.