KC-5722

293T-FZD4-KO-cyno-FZD4-Low Cell Line

59004

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Background of 293T-FZD4-KO-cyno-FZD4-Low Cell Line

FZD4, also known as GPCR. This gene is a member of the frizzled gene family. Members of this family encode seven-transmembrane domain proteins that are receptors for the Wingless type MMTV integration site family of signaling proteins. Most frizzled receptors are coupled to the beta-catenin canonical signaling pathway. This protein may play a role as a positive regulator of the Wingless type MMTV integration site signaling pathway. Research has shown that FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in tumor-associated endothelial cells.

Specifications

Catalog Number	KC-5722
Cell Line Name	293T-FZD4-KO-cyno-FZD4-Low Cell Line
Clone Number	4#
Host Cell Line	293T-FZD4-KO
Description	Stable 293T-FZD4-KO clone expressing exogenous cyno FZD4 gene in low level
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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

Cell Line Generation

293T-FZD4-KO-cyno-FZD4-Low cell line was generated using a lentiviral vector expressing the cyno FZD4 sequence.

Characterization

Figure 1: Characterization of cyno FZD4 overexpression in the 293T-FZD4-KO-cyno-FZD4 stable clone using FACS.

Figure 2: Characterization of cyno FZD4 in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 1μg/mL Puromycin)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 1:4-1:8 every 2-3 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. Bian F, Goda C, Wang G, Lan YW, Deng Z, Gao W, Acharya A, Reza AA, Gomez-Arroyo J, Merjaneh N, Ren X, Goveia J, Carmeliet P, Kalinichenko VV, Kalin TV. FOXF1 promotes tumor vessel normalization and prevents lung cancer progression through FZD4. EMBO Mol Med. 2024 May;16(5):1063-1090. doi: 10.1038/s44321-024-00064-8. Epub 2024 Apr 8. PMID: 38589650; PMCID: PMC11099127.
2. Li CX, Talukder M, Xu YR, Zhu SY, Zhao YX, Li JL. Cadmium aggravates the blood-brain barrier disruption via inhibition of the Wnt7A/β-catenin signaling axis. Environ Pollut. 2023 May 1;324:121400. doi: 10.1016/j.envpol.2023.121400. Epub 2023 Mar 4. PMID: 36878275.