KC-5276

293T-STEAP3-KO Cell Line

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Background of 293T-STEAP3-KO Cell Line

This gene encodes a multipass membrane protein that functions as an iron transporter. The encoded protein can reduce both iron (Fe3+) and copper (Cu2+) cations. STEAP3 contains a functional p53-binding site in its promoter and can be upregulated following p53 activation to enhance cell death in myeloid leukemia cell line and breast cancer cells. Deficiency in this gene can cause anemia. Alternative splicing results in multiple transcript variants.

Specifications

Catalog Number	KC-5276
Cell Line Name	293T-STEAP3-KO Cell Line
Clone Number	2C4
Host Cell Line	293T
Description	Stable 293T clone with human STEAP3 gene knockout, No.2C4
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	90% DMEM+10% FBS
Selection Marker	NA
Morphology	epithelial
Subculture	Split saturated culture 1:5-1:8 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-STEAP3-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-STEAP3-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-STEAP3-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 293T-STEAP3-KO cell line stable clone using Western blot.

Cell Resuscitation

Prewarm culture medium (90% DMEM + 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 1:5-1:8 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

Wang LL, Luo J, He ZH, Liu YQ, Li HG, Xie D, Cai MY. STEAP3 promotes cancer cell proliferation by facilitating nuclear trafficking of EGFR to enhance RAC1-ERK-STAT3 signaling in hepatocellular carcinoma. Cell Death Dis. 2021 Nov 5;12(11):1052. doi: 10.1038/s41419-021-04329-9. PMID: 34741044; PMCID: PMC8571373.
https://www.ncbi.nlm.nih.gov/gene/55240