KC-5548

293T-rat-PSMA Cell Line

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Background of 293T-rat-PSMA Cell Line

FOLH1, also named as prostate-specific membrane antigen (PSMA), is enzyme protein and mainly expressed in prostate epithelium, the proximal tubules of the kidney, the jejunal brush border of the small intestine and ganglia of the nervous system. FOLH1 is overexpressed in human prostate cancer and might be a potential target for tumor therapy. Diseases associated with FOLH1 include Prostate Disease and Hyperhomocysteinemia.

Specifications

Catalog Number	KC-5548
Cell Line Name	293T-rat-PSMA Cell Line
Clone Number	4#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous rat PSMA gene
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-rat-PSMA cell line was generated using a lentiviral vector expressing the rat PSMA sequence.

Characterization

Figure 1: Characterization of rat PSMA overexpression in the 293T-rat-PSMA stable clone using FACS.

Figure 2: Characterization of rat PSMA in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 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. Farolfi A, Calderoni L, Mattana F, Mei R, Telo S, Fanti S, Castellucci P. Current and Emerging Clinical Applications of PSMA PET Diagnostic Imaging for Prostate Cancer. J Nucl Med. 2021 May 10;62(5):596-604. doi: 10.2967/jnumed.120.257238. Epub 2021 Mar 12. PMID: 33712536.
2. O'Keefe DS, Su SL, Bacich DJ, Horiguchi Y, Luo Y, Powell CT, Zandvliet D, Russell PJ, Molloy PL, Nowak NJ, Shows TB, Mullins C, Vonder Haar RA, Fair WR, Heston WD. Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene. Biochim Biophys Acta. 1998 Nov 26;1443(1-2):113-27. doi: 10.1016/s0167-4781(98)00200-0. PMID: 9838072.
3. Bahler CD, Johnson MM, Davicioni E, Zhang J, Cheng L, Green MA, Koch MO. Predictors of Prostate-specific Membrane Antigen (PSMA/FOLH1) Expression in a Genomic Database. Urology. 2020 Oct;144:117-122. doi: 10.1016/j.urology.2020.06.025. Epub 2020 Jun 30. PMID: 32619596.
4. Israeli RS, Powell CT, Fair WR, Heston WD (January 1993). Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen. Cancer Res. 53 (2): 227–30.