KC-3635

4T1-ERBB2-Cell-Line

26703

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Background of 4T1-ERBB2-Cell-Line

ERBB2, also known as CD340. This gene 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. Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized.

Specifications

Catalog Number	KC-3635
Cell Line Name	4T1-ERBB2-Cell-Line
Host Cell Line	4T1
Description	Stable 4T1 cell line expressing exogenous ERBB2 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 4μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:6-1:8 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 12.6 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

4T1 ERBB2 Cell Line was generated using a lentiviral vector expressing the ERBB2 sequence.

Characterization

Figure 1: Characterization of endogenous ERBB2 expression in 4T1 cell using FACS.

Figure 2: Characterization of ERBB2 overexpression in the 4T1 ERBB2 stable clone using FACS.

Figure 3: Characterization of ERBB2 overexpression in 4T1 stable clone using DNA sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 4μg/mL Puromycin)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 1:6-1:8 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

Lee SY, Yoo TK, Kim J, Chung IY, Ko BS, Kim HJ, Lee JW, Son BH, Lee SB. Clinical significance of microinvasive breast cancer across the different subtypes and human epidermal growth factor receptor 2 expression levels. Breast Cancer Res Treat. 2023 Jul;200(1):47-61. doi: 10.1007/s10549-023-06955-7. Epub 2023 May 15. PMID: 37184775.