KC-5364

293T-cyno-MSLN Cell Line

56686

Home » 293T-cyno-MSLN Cell Line

Background of 293T-cyno-MSLN Cell Line

MSLN (Mesothelin) is a Protein Coding gene. Mesothelin is a glycosylphosphatidylinositol-anchored cell-surface protein that may function as a cell adhesion protein. This protein is overexpressed in epithelial mesotheliomas, ovarian cancers and in specific squamous cell carcinomas. Mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. MSLN is considered to play an important role in cell survival, proliferation, and tumor progression. Diseases associated with MSLN include Benign Mesothelioma and Asbestosis.

Specifications

Catalog Number	KC-5364
Cell Line Name	293T-cyno-MSLN Cell Line
Clone Number	5#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno MSLN 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-cyno-MSLN cell line was generated using a lentiviral vector expressing the cyno MSLN sequence.

Characterization

Figure 1: Characterization of cyno MSLN overexpression in the 293T cyno MSLN stable clone using FACS.(Kyinno, Cat#KB-1276)

Figure 2: Characterization of cyno MSLN and its mutants overexpressing in 293T stable clones 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. Li Y, Tian W, Zhang H, Zhang Z, Zhao Q, Chang L, Lei N, Zhang W. MSLN Correlates With Immune Infiltration and Chemoresistance as a Prognostic Biomarker in Ovarian Cancer. Front Oncol. 2022 May 25;12:830570. doi: 10.3389/fonc.2022.830570. PMID: 35692779; PMCID: PMC9174524.
2. Schoutrop E, Poiret T, El-Serafi I, Zhao Y, He R, Moter A, Henriksson J, Hassan M, Magalhaes I, Mattsson J. Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models. J Immunother Cancer. 2023 Feb;11(2):e005691. doi: 10.1136/jitc-2022-005691. PMID: 36746513; PMCID: PMC9906404.
3. Sotoudeh M, Shirvani SI, Merat S, Ahmadbeigi N, Naderi M. MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the potent antigenic targets for CAR T cell therapy of gastric adenocarcinoma. J Cell Biochem. 2019 Apr;120(4):5010-5017. doi: 10.1002/jcb.27776. Epub 2018 Sep 27. PMID: 30260046.