KC-1804

MIAPaCa2-VHL-KO Cell Line

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Background of MIAPaCa2-VHL-KO Cell Line

The von Hippel-Lindau (VHL) gene encodes a tumor suppressor protein that functions as the substrate recognition component of an E3 ubiquitin ligase complex. Its primary role is to target hypoxia-inducible factor alpha subunits (HIF-α) for proteasomal degradation under normoxic conditions. Loss-of-function mutations in VHL disrupt this process, leading to constitutive stabilization of HIF-α and subsequent transcriptional upregulation of hypoxia-responsive genes (e.g., VEGF, PDGF, GLUT1). This drives angiogenesis, cell proliferation, and metabolic adaptation, underpinning the pathogenesis of VHL disease—an autosomal dominant familial cancer syndrome characterized by hemangioblastomas, renal cell carcinomas, and pheochromocytomas. Somatic VHL inactivation is also a hallmark of the majority of sporadic clear cell renal cell carcinomas (ccRCC). Consequently, the VHL-HIF pathway is a major therapeutic target, with drugs inhibiting VEGF signaling (e.g., sunitinib, pazopanib) representing the cornerstone of treatment for advanced RCC. Research continues to explore HIF-2α-specific inhibitors (e.g., belzutifan) and combination strategies.

Specifications

Catalog Number	KC-1804
Cell Line Name	MIAPaCa2-VHL-KO Cell Line
Clone Number	3C2
Host Cell Line	MIAPaCa2
Description	Stable MIAPaCa2 clone with human VHL gene knockout
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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS+2.5%HS
Selection Marker	N/A
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:4 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

MIAPaCa2-VHL-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of MIAPaCa2-VHL-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of MIAPaCa2-VHL-KO cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (DMEM+10% FBS+2.5%HS)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 1:2-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Kaelin, W.G., Jr. (2007). "The von Hippel-Lindau tumor suppressor protein and clear cell renal carcinoma." Clinical Cancer Research, 13(2 Pt 2), 680s-684s.
Gossage, L., et al. (2015). "VHL, the story of a tumour suppressor gene." Nature Reviews Cancer, 15(1), 55-64.
Choueiri, T.K., & Kaelin, W.G., Jr. (2020). "Targeting the HIF2-VEGF axis in renal cell carcinoma." Nature Medicine, 26(10), 1519-1530.