KC-4250

A549-ARID1A-KO-3B2-Cell-Line

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Background of A549-ARID1A-KO-3B2-Cell-Line

The ARID1A gene, encoding the AT-Rich Interaction Domain 1A protein, is a critical component of the mammalian SWI/SNF (switch/sucrose non-fermentable) chromatin remodeling complex. This complex is essential for regulating gene expression by altering chromatin structure, thereby playing a pivotal role in cellular processes such as DNA repair, cell cycle control, and differentiation. ARID1A is characterized by its ability to recognize and bind to AT-rich DNA sequences, facilitating the assembly of the SWI/SNF complex at specific genomic sites.Mutations in ARID1A are associated with a variety of human diseases, most prominently in cancer. It is one of the most frequently mutated genes in ovarian clear cell carcinoma, gastric cancer, and endometrial cancer, often leading to loss of function and contributing to tumor development. These mutations disrupt the SWI/SNF complex, leading to abnormal gene expression patterns that promote tumorigenesis.

Specifications

Catalog Number	KC-4250
Cell Line Name	A549-ARID1A-KO-3B2-Cell-Line
Host Cell Line	A549
Description	Stable A549 clone with human ARID1A gene knockout, No.3B
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

A549-ARID1A-KO-3B2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of A549-ARID1A-KO-3B2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of A549-ARID1A-KO-3B2 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

Berger, S. L., et al. (2009). The landscape of histone modifications across 1% of the human genome in five human cell lines.Genome Research, 19(6), 1190-1206. https://doi.org/10.1101/gr.083637.108
Yamaguchi, H., et al. (2012). Frequent inactivating mutations of ARID1A in gastric cancers.Nature Genetics, 44(3), 302-305. https://doi.org/10.1038/ng.1092
Carvalho, C., et al. (2014). Germline mutations in ARID1A cause a syndromic form of intellectual disability with craniofacial and skeletal anomalies.American Journal of Human Genetics, 94(6), 838-847. https://doi.org/10.1016/j.ajhg.2014.04.008