KC-5861

293T-rat-RAMP3-CALCR Cell Line

60111

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Background of 293T-rat-RAMP3-CALCR Cell Line

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. Diseases associated with RAMP3 include Immunodeficiency 53 and Immunodeficiency 57 With Autoinflammation.
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-5861
Cell Line Name	293T-rat-RAMP3-CALCR Cell Line
NCBI/UniProt Accession Number	rat-RAMP3: NM_020100.2/rat-CALCR: P32214-2
Clone Number	6#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous rat RAMP3 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 1:4-1:5 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-rat-RAMP3-CALCR cell line was generated using a lentiviral vector expressing the rat RAMP3 CALCR sequence.

Characterization

Figure 1: Characterization of rat CALCR overexpression in the 293T-rat-RAMP3-CALCR stable clone using FACS.

Figure 2: Characterization of rat RAMP3 overexpression in the293T-rat-RAMP3-CALCR stable clone using QPCR.

Figure 3: Characterization of rat RAMP3 CALCR in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 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 1:4-1:5 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. Yang L, Fang A, Zhou S, Liu H. -RAMP3 promotes hepatocellular carcinoma tumor cell-mediated CCL2 degradation by supporting membrane distribution of ACKR2. Int Immunopharmacol. 2024 Dec 25;143(Pt 2):113419. doi: 10.1016/j.intimp.2024.113419. Epub 2024 Oct 21. PMID: 39437486.
2. Hou Y, Sun L, LaFleur MW, Huang L, Lambden C, Thakore PI, Geiger-Schuller K, Kimura K, Yan L, Zang Y, Tang R, Shi J, Barilla R, Deng L, Subramanian A, Wallrapp A, Choi HS, Kye YC, Ashenberg O, Schiebinger G, Doench JG, Chiu IM, Regev A, Sharpe AH, Kuchroo VK. Neuropeptide signalling orchestrates T cell differentiation. Nature. 2024 Nov;635(8038):444-452. doi: 10.1038/s41586-024-08049-w. Epub 2024 Oct 16. PMID: 39415015; PMCID: PMC11951087.
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.