KC-3208

Ba/F3-ERBB2-A775-G776insYVMA-C805S-Cell-Line

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Background of Ba/F3-ERBB2-A775-G776insYVMA-C805S-Cell-Line

ERBB2 encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein has no ligand binding domain of its own and therefore cannot bind growth factors. However, it does bind tightly to other ligand-bound EGF receptor family members to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signalling pathways, such as those involving mitogen-activated protein kinase and phosphatidylinositol-3 kinase. Allelic variations at amino acid positions 654 and 655 of isoform a (positions 624 and 625 of isoform b) have been reported, with the most common allele, Ile654/Ile655, shown here. Amplification and/or overexpression of this gene has been reported in numerous cancers, including breast and ovarian tumors. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-3208
Cell Line Name	Ba/F3-ERBB2-A775-G776insYVMA-C805S-Cell-Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous ERBB2 gene bearing A775-G776insYVMA-C805S mutation
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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS
Selection Marker	Puromycin
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3 ERBB2-A775-G776insYVMA-C805S cell Line was generated using retrovirus vector expressing human ERBB2-A775-G776insYVMA-C805S sequence.

Characterization

Figure 1: Characterization of ERBB2 mutation in the Ba/F3 stable clone using PCR sequencing.

Figure 1: 1. Harvest and seed the Ba/F3 cells expressing ERBB2 mutant in 96-well plate (3000 cells/90μL medium). 2. Next day, add 10μL 10× serially diluted compound solution each well and incubate the plates for another 72 hours. 3. Add 100μL Cell Titer-Glo each well, mixed and readout using Envision. 4. Plot the dose-responsive curve and fit the IC₅₀ (the centration of 50% inhibition of DMSO vehicle treated clones)using GraphPad Prism software (Version 5).

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 1:10 every 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

Subramanian J, Katta A, Masood A, Vudem DR, Kancha RK. Emergence of ERBB2 Mutation as a Biomarker and an Actionable Target in Solid Cancers. Oncologist. 2019 Dec;24(12):e1303-e1314. doi: 10.1634/theoncologist.2018-0845. Epub 2019 Jul 10. PMID: 31292270; PMCID: PMC6975965.
Waliany S, Wakelee H, Ramchandran K, Das M, Huang J, Myall N, Li C, Pagtama J, Tisch AH, Neal JW. Characterization of ERBB2 (HER2) Alterations in Metastatic Non-small Cell Lung Cancer and Comparison of Outcomes of Different Trastuzumab-based Regimens. Clin Lung Cancer. 2022 Sep;23(6):498-509. doi: 10.1016/j.cllc.2022.05.015. Epub 2022 Jun 3. PMID: 35753988.