KC-6269

293T-NFκB-Luc2-TNFRSF1A-KO Cell Line

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Background of 293T-NFκB-Luc2-TNFRSF1A-KO Cell Line

TNFRSF1A, officially named TNF receptor superfamily member 1A, is a protein-coding gene located on human chromosome 12p13.31. It encodes tumor necrosis factor receptor 1 (TNFR1), which exists in both membrane-bound and soluble forms and specifically binds to tumor necrosis factor alpha (TNFα). Binding of membrane-bound TNFα to TNFR1 induces receptor trimerization, activating signaling pathways that regulate cell survival, apoptosis, and inflammation. The soluble form of TNFR1, generated by proteolytic processing, inhibits inflammation by sequestering free TNFα. Mutations in TNFRSF1A are causatively linked to tumor necrosis factor receptor-associated periodic syndrome (TRAPS) and may be associated with multiple sclerosis. This gene is ubiquitously expressed in various human tissues, underscoring its critical role in immune homeostasis.

Specifications

Catalog Number	KC-6269
Cell Line Name	293T-NFκB-Luc2-TNFRSF1A-KO Cell Line
NCBI/UniProt Accession Number	7132
Clone Number	2A3
Host Cell Line	293T-NFκB-Luc2
Description	Stable 293T-NFκB-Luc2 cell line with TNFRSF1A gene knockout, No.2A3
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+150μg/mL Hygromycin B
Selection Marker	NA
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-NFκB-Luc2-TNFRSF1A-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-NFκB-Luc2-TNFRSF1A-KO Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-NFκB-Luc2-TNFRSF1A-KO Cell Line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 293T-NFκB-Luc2(Left) and 293T-NFκB-Luc2-TNFRSF1A-KO(Right) Cell Line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 150μg/mL Hygromycin B) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Galon J, Aksentijevich I, McDermott MF, O'Shea JJ, Kastner DL. TNFRSF1A mutations and autoinflammatory syndromes. Curr Opin Immunol. 2000 Aug;12(4):479-86. doi: 10.1016/s0952-7915(00)00124-2. PMID: 10899034.
Aksentijevich I, Galon J, Soares M, Mansfield E, Hull K, Oh HH, Goldbach-Mansky R, Dean J, Athreya B, Reginato AJ, Henrickson M, Pons-Estel B, O'Shea JJ, Kastner DL. The tumor-necrosis-factor receptor-associated periodic syndrome: new mutations in TNFRSF1A, ancestral origins, genotype-phenotype studies, and evidence for further genetic heterogeneity of periodic fevers. Am J Hum Genet. 2001 Aug;69(2):301-14. doi: 10.1086/321976. Epub 2001 Jul 6. Erratum in: Am J Hum Genet 2001 Nov;69(5):1160. PMID: 11443543; PMCID: PMC1235304.
Gerritsma AM, Sutera D, Cantarini L, Cattalini M, Lachmann HJ, Minden K, Jansson AF, Touitou I, Bustaffa M, Antón J, Insalaco A, Moreno E, Sanchez-Manubens J, Ruperto N, Frenkel J, Gattorno M; for Eurofever/Eurotraps projects and Paediatric Rheumatology International Trials Organisation (PRINTO). TNFRSF1A-pR92Q variant identifies a subset of patients more similar to systemic undifferentiated recurrent fever than TNF receptor-associated periodic syndrome. Clin Exp Rheumatol. 2023 Oct;41(10):1998-2007. doi: 10.55563/clinexprheumatol/am4phc. Epub 2023 Jul 11. PMID: 37470237.
Zhao J, Yang X, Zhang H, Gu X. Identification of TNFRSF1A as a novel regulator of carfilzomib resistance in multiple myeloma. Oncol Res. 2023 Dec 28;32(2):325-337. doi: 10.32604/or.2023.030770. PMID: 38186567; PMCID: PMC10765120.
Acuna K, Choudhary A, Locatelli E, Rodriguez DA, Martin ER, Levitt RC, Galor A. Impact of Tumor Necrosis Factor Receptor 1 (TNFR1) Polymorphism on Dry Eye Disease. Biomolecules. 2023 Jan 31;13(2):262. doi: 10.3390/biom13020262. PMID: 36830631; PMCID: PMC9953194.