KC-5536

KCL22-BCR-ABL-T315I-KI Cell Line

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Background of KCL22-BCR-ABL-T315I-KI Cell Line

A reciprocal translocation between chromosomes 22 and 9 produces the Philadelphia chromosome, which is often found in patients with chronic myelogenous leukemia. The chromosome 22 breakpoint for this translocation is located within the BCR gene. The translocation produces a fusion protein which is encoded by sequence from both BCR and ABL, the gene at the chromosome 9 breakpoint. BCR-ABL and its mutants can promote and maintain the malignant behavior of the cancer cells. The identification of BCR-ABL as a driver gene has led to the rapid development of anticancer therapeutics agents, including Imatinib, Dasatinib, Ponatinib and Nilotinib.

Specifications

Catalog Number	KC-5536
Cell Line Name	KCL22-BCR-ABL-T315I-KI Cell Line
Clone Number	2A3
Host Cell Line	KCL22
Description	Stable KCL22 clone expressing endogenous BCR-ABL gene bearing T315I mutations, 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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	lymphoblast-like
Subculture	Split saturated culture 1:5-1:8 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 24 hours
Mycoplasma Status	Negative

Cell Line Generation

KCL22-BCR-ABL-T315I-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of KCL22-BCR-ABL-T315I-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of KCL22-BCR-ABL-T315I-KI cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of Dose-response curves and IC50 values for KCL22 and KCL22-BCR-ABL-T315I-KI cells treated with Dasatinib,Ponatinib,Imatinib,Nilotinib and Asciminib over 3 days.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 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 1:5-1:8 every 2-3 days; seed out at about 1-3 ×10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Weisberg, E., Manley, P. W., Cowan-Jacob, S. W., Hochhaus, A. & Griffin, J. D. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nat Rev Cancer 7, 345ÿ356 (2007).