KC-4170

A549-SETD2-KO-2A3-Cell-Line

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Background of A549-SETD2-KO-2A3-Cell-Line

The SETD2 gene, encoding the SET domain containing 2 protein, is a key player in the epigenetic regulation of gene expression. This enzyme specifically methylates lysine 36 on histone H3 (H3K36me3), a modification that is critical for the proper transcriptional elongation and termination of genes, as well as for the maintenance of genomic stability. Mutations or dysregulation of SETD2 have been linked to various diseases, most notably cancers, where alterations in H3K36 methylation patterns can lead to oncogene activation and tumor suppressor gene silencing.Research has also highlighted the role of SETD2 in DNA damage response pathways and RNA processing, underscoring its multifaceted impact on cellular homeostasis. In renal cell carcinoma and other malignancies, loss-of-function mutations in SETD2 correlate with poor prognosis, suggesting its potential as a biomarker and therapeutic target.

Specifications

Catalog Number	KC-4170
Cell Line Name	A549-SETD2-KO-2A3-Cell-Line
Host Cell Line	A549
Description	Stable A549 clone with human SETD2 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	McCoy5A+20% FBS+10% DMSO
Propagation Medium	McCoy5A+10% FBS
Selection Marker	N/A
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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
In Vivo Validation	NA

Cell Line Generation

Characterization

Figure 1: Characterization of A549-SETD2-KO-2A3 cell line stable clone using PCR sequencing.

Figure 2: Characterization of A549-SETD2-KO-2A3 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of A549-SETD2-KO-2A3 cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (McCoy5A + 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 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% McCoy5A + 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

Fang, J., & Zhang, Y. (2014). Histone H3 lysine 36 methylation: an emerging regulator of gene expression and genome stability.Nature Reviews Molecular Cell Biology, 15(6), 371-383. https://doi.org/10.1038/nrm3803
Couture, J. B., & Shilatifard, A. (2016). Histone H3K36 methylation: a double-edged sword for cancer.Nature Reviews Cancer, 16(12), 788-802. https://doi.org/10.1038/nrc.2016.109
Fiorino, E., et al. (2019). SETD2 and H3K36me3: Implications for cancer and beyond.Journal of Cellular Physiology, 234(6), 7841-7852. https://doi.org/10.1002/jcp.27156