The VHL gene on chromosome 3 encodes the von Hippel-Lindau (VHL) protein, which plays a critical role in tumor suppression and the development of Von Hippel-Lindau syndrome (VHL). This tumor suppressor gene regulates the stability of hypoxia-inducible factors (HIFs), transcription factors involved in cellular responses to low oxygen levels. The VHL protein targets hydroxylated HIFs for degradation, ensuring their tight regulation. Mutations in the VHL gene disrupt this process, accumulating HIFs and the subsequent upregulation of HIF target genes involved in angiogenesis and cell proliferation. Individuals with VHL syndrome have an increased risk of developing various tumors, including hemangioblastomas, renal cell carcinomas, pheochromocytomas, and pancreatic cysts. Understanding the VHL gene and its mutations has important implications for developing targeted therapies and early detection strategies for VHL-associated tumors.
Specifications
Catalog Number:
KC-1804
Cell Line Name:
MIAPaCa2 VHL KO 3C2 Cell Line
Host Cell Line:
Human MIAPaCa2 cell line
Description:
Miapaca2 cell line endogenous VHL gene knockout
Quantity:
One vial of frozen cells (5×106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% DMEM+20% FBS+10% DMSO
Propagation Medium:
DMEM+10%FBS+2.5%HS
Selection Marker:
Morphology:
Epithelial
Subculture:
Split saturated culture 1:3-1:6 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 48 hours
Mycoplasma Status:
Negative
Cell Line Generation
MIAPACA2-VHL-KO Cell Line was generated using the CRISPR-Cas9 method
Characterization
Application
Cell Resuscitation
1. Prewarm culture medium (DMEM + 10% FBS + 2.5% HS)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL.
Cell Freezing
1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage
References
1. Latif F, et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science. 1993; 260(5112):1317-1320.
2. Kaelin WG Jr. Molecular basis of the VHL hereditary cancer syndrome. Nat Rev Cancer. 2002; 2(9):673-682.
3. Gossage L, et al. Clinical and genetic analysis of von Hippel-Lindau syndrome-associated pancreatic neuroendocrine tumors. Clin Cancer Res. 2015; 21(24):5542-5550.
4. Maher ER, et al. Von Hippel-Lindau disease: a clinical and scientific review. Eur J Hum Genet. 2018; 26(1):13-29.
