KC-5322

HepG2-GPC3-KO Cell Line

55492

Home » HepG2-GPC3-KO Cell Line

Background of HepG2-GPC3-KO Cell Line

The Glypican-3 (GPC3) gene encodes a heparan sulfate proteoglycan anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) moiety. GPC3 is highly expressed during embryonic development but shows restricted expression in adult tissues. It plays critical roles in cell proliferation, differentiation, and apoptosis by modulating signaling pathways such as Wnt, Hedgehog, and YAP. GPC3 is implicated in cancer, particularly hepatocellular carcinoma (HCC), where it is overexpressed and serves as a diagnostic and prognostic biomarker. Additionally, GPC3 mutations are associated with Simpson-Golabi-Behmel syndrome (SGBS), an X-linked overgrowth disorder characterized by developmental abnormalities.

Specifications

Catalog Number	KC-5322
Cell Line Name	HepG2-GPC3-KO Cell Line
Clone Number	1A2
Host Cell Line	HepG2
Description	Stable HepG2 cell line with GPC3 gene knockout, No.1A2
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	70% MEM+20% FBS+10% DMSO
Propagation Medium	MEM+10% FBS+0.01mM NEAA
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 1-3 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 25.65 hours
Mycoplasma Status	Negative

Cell Line Generation

HepG2-GPC3-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of HepG2-GPC3-KO Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of HepG2-GPC3-KO Cell Line stable clone using RT-PCR sequencing.

Figure 3: Characterization of HepG2-GPC3-KO Cell Line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (MEM + 10% FBS + 0.01mM NEAA) 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 1:4-1:8 every 1-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% MEM + 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

Filmus J, Capurro M. Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma. FEBS J. 2013 May;280(10):2471-6. doi: 10.1111/febs.12126. Epub 2013 Jan 31. PMID: 23305321.
Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. 2003 Jul;125(1):89-97. doi: 10.1016/s0016-5085(03)00689-9. PMID: 12851874.
Pilia, G., Hughes-Benzie, R., MacKenzie, A. et al. Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome. Nat Genet 12, 241–247 (1996). https://doi.org/10.1038/ng0396-241