KC-4168

A549-PBRM1-KO-2A4-Cell-Line

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Background of A549-PBRM1-KO-2A4-Cell-Line

The PBRM1 gene, which stands for Polybromo 1, encodes a subunit of the SWI/SNF (Switch/Sucrose Non-Fermentable) chromatin remodeling complex. This complex plays a critical role in maintaining normal chromatin structure and regulating gene expression. The encoded protein contains a bromodomain, which allows it to bind acetylated histones, and a bromo-adjacent homology (BAH) domain, which is involved in DNA binding and chromatin targeting.Mutations in PBRM1 are frequently observed in several types of cancer, most notably in clear cell renal cell carcinoma (ccRCC), where it is one of the most commonly mutated genes. Loss-of-function mutations in PBRM1 have been associated with impaired chromatin remodeling, leading to aberrant gene expression and subsequent tumorigenesis. Furthermore, PBRM1 mutations have been correlated with better prognosis in ccRCC patients, indicating its potential as a predictive biomarker.

Specifications

Catalog Number	KC-4168
Cell Line Name	A549-PBRM1-KO-2A4-Cell-Line
Host Cell Line	A549
Description	Stable A549 clone with human PBRM1 gene knockout, No.2A4
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-PBRM1-KO-2A4 cell line stable clone using PCR sequencing.

Figure 2: Characterization of A549-PBRM1-KO-2A4 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of A549-PBRM1-KO-2A4 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

Vogelstein, B., Papadopoulos, N., Velculescu, V. E., Zhou, S., Diaz, L. A., Jr., & Kinzler, K. W. (2013). Cancer genome landscapes.Science, 339(6127), 1546-1558. https://doi.org/10.1126/science.1235122
Vogelstein, B., Papadopoulos, N., Velculescu, V. E., Zhou, S., Diaz, L. A., Jr., & Kinzler, K. W. (2013). Cancer genome landscapes.Science, 339(6127), 1546-1558. https://doi.org/10.1126/science.1235122
Wang, Z., et al. (2016). PBRM1 mutations are associated with favorable prognosis and distinct immune microenvironment in clear cell renal cell carcinoma.Clinical Cancer Research, 22(12), 2889-2900. https://doi.org/10.1158/1078-0432.CCR-15-2765