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KC-5108

COS7-CRE-Luc2-CALCR-RAMP3 Cell Line

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Home » COS7-CRE-Luc2-CALCR-RAMP3 Cell Line

Background of COS7-CRE-Luc2-CALCR-RAMP3 Cell Line

CRE-Luc2 is a CRE response element-based luciferase reporter system for monitoring intracellular cAMP signaling activity, widely used in GPCR functional screening and signal transduction studies.CALCR (Calcitonin Receptor) is a Protein Coding gene.This gene encodes a high affinity receptor for the peptide hormone calcitonin and belongs to a subfamily of seven transmembrane-spanning G protein-coupled receptors. The encoded protein is involved in maintaining calcium homeostasis and in regulating osteoclast-mediated bone resorption. Polymorphisms in this gene have been associated with variations in bone mineral density and onset of osteoporosis. Alternate splicing results in multiple transcript variants. RAMP3 (Receptor Activity Modifying Protein 3) is a Protein Coding gene. The protein encoded by this gene is a member of the RAMP family of single-transmembrane-domain proteins, called receptor (calcitonin) activity modifying proteins (RAMPs). RAMPs are type I transmembrane proteins with an extracellular N terminus and a cytoplasmic C terminus. RAMPs are required to transport calcitonin-receptor-like receptor (CRLR) to the plasma membrane. CRLR, a receptor with seven transmembrane domains, can function as either a calcitonin-gene-related peptide (CGRP) receptor or an adrenomedullin receptor, depending on which members of the RAMP family are expressed. In the presence of this (RAMP3) protein, CRLR functions as an adrenomedullin receptor.

Specifications

Catalog NumberKC-5108
Cell Line NameCOS7-CRE-Luc2-CALCR-RAMP3 Cell Line
NCBI/UniProt Accession NumberNM_001742.4, NM_005856.3
Clone Number11#
Host Cell LineCOS7
DescriptionStable overexpression of the CALCR and RAMP3 gene in COS7 cells stably expressing an exogenous luciferase gene driven by the CRE response element.
QuantityOne vial of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% DMEM + 20% FBS + 10% DMSO
Propagation MediumDMEM+10%FBS+4 µg/mL puromycin+500 µg/mL Hygromycin B
Selection MarkerPuromycin | Hygromycin B
MorphologyEpithelial
SubcultureSplit saturated culture 1:4-1:5 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

Cell Line Generation

COS7-CRE-Luc2-CALCR-RAMP3 was generated by stable overexpression of the CALCR and RAMP3 gene in COS7 cells stably expressing an exogenous luciferase gene driven by the CRE response element.

Characterization

Figure1: Characterization of CALCR overexpressing in COS7-CRE-Luc2 stable clones using FACS.

Figure2: Characterization of RAMP3 overexpressing in COS7-CRE-Luc2 stable clones using qPCR.

Figure3: A total of 20,000 293T-CRE-Luc2-CALCR-RAMP3 cells were seeded into a 96-well plate, followed by serum starvation for 6 hours, treatment with different concentrations of calcitonin for 16 hours, and signal detection using the Bright-Glo system.

Figure4: Characterization of COS7-CRE-Luc2-CALCR-RAMP3 cell line stable clone using PCR sequencing.

Cell Resuscitation

  1. Prewarm culture medium (DMEM+10%FBS+4 µg/mL puromycin+500 µg/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 1:4-1:5 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. Tsai FJ, Chen WC, Chen HY, Tsai CH. The ALUI calcitonin receptor gene polymorphism (TT) is associated with low bone mineral density and susceptibility to osteoporosis in postmenopausal women. Gynecol Obstet Invest. 2003;55(2):82-7. doi: 10.1159/000070179. PMID: 12771454.
  2. Wookey PJ, McLean CA, Hwang P, Furness SG, Nguyen S, Kourakis A, Hare DL, Rosenfeld JV. The expression of calcitonin receptor detected in malignant cells of the brain tumour glioblastoma multiforme and functional properties in the cell line A172. Histopathology. 2012 May;60(6):895-910. doi: 10.1111/j.1365-2559.2011.04146.x. Epub 2012 Feb 15. PMID: 22335784.
  3. Flahaut M, Pfister C, Rossier BC, Firsov D. N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor. Biochemistry. 2003 Sep 2;42(34):10333-41. doi: 10.1021/bi0347508. PMID: 12939163.

Use License Agreement

Research Use Only.
Not for use in diagnostic procedures or therapeutic applications.
Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.

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