KC-5955

293T-cyno-GPNMB Cell Line

64004

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Background of 293T-cyno-GPNMB Cell Line

GPNMB (Glycoprotein Nmb) is a Protein Coding gene. The protein encoded by this gene is a type I transmembrane glycoprotein which shows homology to the pMEL17 precursor, a melanocyte-specific protein. GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. GPNMB shows expression in the lowly metastatic human melanoma cell lines and xenografts but does not show expression in the highly metastatic cell lines. Diseases associated with GPNMB include Amyloidosis, Primary Localized Cutaneous, 3 and Lichen Amyloidosis.

Specifications

Catalog Number	KC-5955
Cell Line Name	293T-cyno-GPNMB Cell Line
NCBI/UniProt Accession Number	XM_045388918.2
Clone Number	6#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno GPNMB gene
Quantity	One vial of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:5 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-cyno-GPNMB cell line was generated using a lentiviral vector expressing the cyno GPNMB sequence.

Characterization

Figure 1: Characterization of endogenous cyno GPNMB expression in 293T cells using FACS.

Figure 2: Characterization of cyno GPNMB overexpression in the 293T-cyno-GPNMB stable clone using FACS.

Figure 3: Characterization of cyno GPNMB in the 293T-cyno-GPNMB stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (DMEM+10% FBS +1μg/mL Puromycin) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:5 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Saade M, Araujo de Souza G, Scavone C, Kinoshita PF. The Role of GPNMB in Inflammation. Front Immunol. 2021 May 12;12:674739. doi: 10.3389/fimmu.2021.674739. PMID: 34054862; PMCID: PMC8149902.
2. Liguori M, Digifico E, Vacchini A, Avigni R, Colombo FS, Borroni EM, Farina FM, Milanesi S, Castagna A, Mannarino L, Craparotta I, Marchini S, Erba E, Panini N, Tamborini M, Rimoldi V, Allavena P, Belgiovine C. The soluble glycoprotein NMB (GPNMB) produced by macrophages induces cancer stemness and metastasis via CD44 and IL-33. Cell Mol Immunol. 2021 Mar;18(3):711-722. doi: 10.1038/s41423-020-0501-0. Epub 2020 Jul 29. PMID: 32728200; PMCID: PMC8027814.
3. Neal ML, Boyle AM, Budge KM, Safadi FF, Richardson JR. The glycoprotein GPNMB attenuates astrocyte inflammatory responses through the CD44 receptor. J Neuroinflammation. 2018 Mar 8;15(1):73. doi: 10.1186/s12974-018-1100-1. PMID: 29519253; PMCID: PMC5842560.

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.