KC-6479

293T-cyno-DR3 Cell Line

62458

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Background of 293T-cyno-DR3 Cell Line

DR3 (Death Receptor 3) , officially named TNFRSF25 (TNF Receptor Superfamily Member 25), is also known as APO-3, TRAMP, LARD, and WSL-1. It is a death domain-containing receptor primarily expressed in lymphocyte-rich tissues such as spleen, thymus, and colon. Upon binding its ligand TL1A (TNFSF15), DR3 regulates both apoptosis and NF-κB signaling, playing a critical role in T-cell homeostasis and immune regulation. DR3 is implicated in autoimmune diseases including inflammatory bowel disease (IBD) and rheumatoid arthritis, and exhibits dual roles in cancer, acting as both a tumor suppressor and pro-metastatic factor depending on context. While no direct-targeting drugs are clinically approved, DR3 agonists are being explored preclinically for cancer immunotherapy, and the TL1A/DR3 axis remains an active therapeutic target for chronic inflammatory conditions.

Specifications

Catalog Number	KC-6479
Cell Line Name	293T-cyno-DR3 Cell Line
NCBI/UniProt Accession Number	XM_015443331.2
Clone Number	1#
Host Cell Line	293T cell line
Description	Stable 293T cell line expressing exogenous cyno DR3 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% basal medium+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
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-cyno-DR3 cell line was generated using a lentiviral vector expressing the cyno DR3 sequence.

Characterization

Figure 1: Characterization of cyno-DR3 overexpression in the 293T-cyno-DR3 stable clone using FACS.

Figure 2: Characterization of cyno-DR3 in the 293T-cyno-DR3 stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) 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 1:4-1:8 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. Valatas, Vassilis et al. “TL1A (TNFSF15) and DR3 (TNFRSF25): A Co-stimulatory System of Cytokines With Diverse Functions in Gut Mucosal Immunity.” Frontiers in immunology vol. 10 583. 27 Mar. 2019, doi:10.3389/fimmu.2019.00583.
2. Shimodaira, Yosuke et al. “DR3 Regulates Intestinal Epithelial Homeostasis and Regeneration After Intestinal Barrier Injury.” Cellular and molecular gastroenterology and hepatology vol. 16,1 (2023): 83-105. doi:10.1016/j.jcmgh.2023.03.008.
3. Li, Jingyu et al. “Activation of DR3 signaling causes loss of ILC3s and exacerbates intestinal inflammation.” Nature communications vol. 10,1 3371. 29 Jul. 2019, doi:10.1038/s41467-019-11304-8.
4. Jacob, Noam et al. “Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo.” Scientific reports vol. 10,1 18189. 23 Oct. 2020, doi:10.1038/s41598-020-75168-5.
5. Liman, Nurcin et al. “The ever-expanding role of cytokine receptor DR3 in T cells.” Cytokine vol. 176 (2024): 156540. doi:10.1016/j.cyto.2024.156540.