KC-5145

293T-RAMP2-CALCR Cell Line

56065

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Background of 293T-RAMP2-CALCR Cell Line

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). Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. 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 (RAMP2) protein, CRLR functions as an adrenomedullin receptor.
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 calcitonin receptor (CALCR) is an essential protein for maintaining calcium homeostasis and has been reported to be upregulated in numerous cancers. 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.

Specifications

Catalog Number	KC-5145
Cell Line Name	293T-RAMP2-CALCR Cell Line
Clone Number	9-13#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human RAMP2 and CALCR 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

Cell Line Generation

293T-RAMP2-CALCR cell line was generated using a lentiviral vector expressing the human RAMP2 and CALCR sequence.

Characterization

Figure 1: Characterization of RAMP2 and CALCR overexpression in the 293T stable clone using FACS.

Figure 2: Characterization of RAMP2 and CALCR in the 293T stable clone using PCR sequencing.

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. Krishna Kumar K, O\'Brien ES, Habrian CH, Latorraca NR, Wang H, Tuneew I, Montabana E, Marqusee S, Hilger D, Isacoff EY, Mathiesen JM, Kobilka BK. Negative allosteric modulation of the glucagon receptor by RAMP2. Cell. 2023 Mar 30;186(7):1465-1477.e18. doi: 10.1016/j.cell.2023.02.028. PMID: 37001505; PMCID: PMC10144504.
2. Ichikawa-Shindo Y, Sakurai T, Kamiyoshi A, Kawate H, Iinuma N, Yoshizawa T, Koyama T, Fukuchi J, Iimuro S, Moriyama N, Kawakami H, Murata T, Kangawa K, Nagai R, Shindo T. The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity. J Clin Invest. 2008 Jan;118(1):29-39. doi: 10.1172/JCI33022. PMID: 18097473; PMCID: PMC2147670.
3.Yan H, Xing Z, Liu S, Gao P, Wang Q, Guo G. CALCR exacerbates renal cell carcinoma progression via stabilizing CD44. Aging (Albany NY). 2024 Jul 9;16(13):10765-10783. doi: 10.18632/aging.205586. Epub 2024 Jul 9. PMID: 38985127; PMCID: PMC11272109.
4.He T, Ling F. CALCR knockdown inhibits the development and progression of non-small-cell lung cancer. Carcinogenesis. 2021 Nov 12;42(11):1390-1398. doi: 10.1093/carcin/bgab076. PMID: 34417812.