KC-4527

TMD8-BTK-C481F-KI Cell Line

47759

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Background of TMD8-BTK-C481F-KI Cell Line

The Bruton's tyrosine kinase (BTK) gene encodes a non-receptor tyrosine kinase that plays a critical role in B-cell development, activation, and signaling. Mutations in BTK can lead to impaired B-cell function and are associated with various hematologic malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). One of the most clinically significant mutations is the C481F substitution in the BTK protein. This mutation occurs at cysteine residue 481, which is located within the ATP-binding pocket of BTK. The C481F mutation confers resistance to ibrutinib, a potent and selective inhibitor of BTK widely used in the treatment of B-cell malignancies. This resistance arises because the mutant cysteine is replaced by phenylalanine, disrupting the covalent binding between ibrutinib and BTK. Consequently, patients harboring this mutation may experience disease progression despite ibrutinib therapy. Understanding the molecular mechanisms underlying this resistance is crucial for developing alternative therapeutic strategies.

Specifications

Catalog Number	KC-4527
Cell Line Name	TMD8-BTK-C481F-KI Cell Line
Host Cell Line	TMD8
Description	Stable TMD8 cell line with endogenous BTK-C481F knock-in
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% RPMI-1640+20% FBS+10% DMSO
Propagation Medium	RPMI-1640+10% FBS
Selection Marker	NA
Morphology	lymphoblast
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

TMD8-BTK-C481F-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of TMD8-BTK-C481F-KI Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of TMD8-BTK-C481F-KI Cell Line stable clone using RT PCR sequencing.

Figure 3: Characterization of Dose-response curves and IC50 values for TMD8 and TMD8-BTK-C481F-KI cells treated with Ibrutinib,Pirtobrutinib,Zanubrutinib,Orelabrutinib,BGB-16673 and NX-5948 over 5 days.

Cell Resuscitation

Prewarm culture medium (RPMI-1640 + 10% FBS)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% RPMI-1640 + 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

Woyach JA, Furman RR, Liu TM, Ozer HG, Zapatka M, Ruppert AS, Xue L, Li DH, Steggerda SM, Versele M, Dave SS, Zhang J, Yilmaz AS, Jaglowski SM, Blum KA, Lozanski A, Lozanski G, James DF, Barrientos JC, Lichter P, Stilgenbauer S, Buggy JJ, Chang BY, Johnson AJ, Byrd JC. Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014 Jun 12;370(24):2286-94. doi: 10.1056/NEJMoa1400029. Epub 2014 May 28. PMID: 24869598; PMCID: PMC4144824.
2.Dhami K, Chakraborty A, Gururaja TL, Cheung LW, Sun C, DeAnda F, Huang X. Kinase-deficient BTK mutants confer ibrutinib resistance through activation of the kinase HCK. Sci Signal. 2022 May 31;15(736):eabg5216. doi: 10.1126/scisignal.abg5216. Epub 2022 May 31. PMID: 35639855.
3.Mehra S, Nicholls M, Taylor J. The Evolving Role of Bruton's Tyrosine Kinase Inhibitors in B Cell Lymphomas. Int J Mol Sci. 2024 Jul 9;25(14):7516. doi: 10.3390/ijms25147516. PMID: 39062757; PMCID: PMC11276629.