KC-5256

MV-4-11-GSPT1-G575N-KI Cell Line

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Background of MV-4-11-GSPT1-G575N-KI Cell Line

The GSPT1 gene, encoding the G1 to S phase transition 1 protein (also known as eukaryotic release factor 3a or eRF3a), is a crucial component of the translation termination machinery in eukaryotic cells. GSPT1, along with eRF1, forms the eukaryotic release factor complex that recognizes stop codons on messenger RNA (mRNA) and facilitates the release of the newly synthesized polypeptide from the ribosome, thereby terminating protein synthesis. This process is essential for the accurate and efficient translation of genetic information.Recent studies have highlighted the involvement of GSPT1 in various diseases, particularly in cancer. Overexpression of GSPT1 has been observed in several types of cancer, including lung, breast, and colorectal cancers, and is associated with poor prognosis. Inhibition of GSPT1 has been shown to induce apoptosis and reduce the viability of cancer cells, suggesting its potential as a therapeutic target. Additionally, GSPT1 has been implicated in the regulation of cellular senescence and the maintenance of stem cell properties.Understanding the multifaceted roles of GSPT1 in translation termination and other cellular processes is essential for elucidating its contributions to disease pathogenesis and for developing targeted therapeutic strategies.

Specifications

Catalog Number	KC-5256
Cell Line Name	MV-4-11-GSPT1-G575N-KI Cell Line
Clone Number	2B1
Host Cell Line	MV-4-11
Description	Stable MV-4-11 clone expressing exogenous GSPT1 gene bearing G575N mutations, No.2B1
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS
Selection Marker	NA
Morphology	lymphoblast
Subculture	Split saturated culture 1:4-1:5 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

MV-4-11-GSPT1-G575N-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of MV-4-11-GSPT1-G575N-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of MV-4-11-GSPT1-G575N-KI cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of dose-response curves for GSPT1 inhibitors on MV-4-11 and MV-4-11-GSPT1-G575N-KI cells.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Ivanov, I. P., & Atkins, J. F. (2000). Ribosome Stalling and Peptide Release during Translational Termination.Molecular Cell, 6(5), 973-984. https://doi.org/10.1016/S1097-2765(00)00119-0.
Inada, T., & Aiba, H. (2005). Mechanisms Regulating Translational Termination in Eukaryotes.Progress in Nucleic Acid Research and Molecular Biology, 79, 1-47. https://doi.org/10.1016/S0079-6603(05)79001-8.
Wang, L., et al. (2019). GSPT1 regulates cell cycle progression and response to DNA damage.Cell Death & Differentiation, 26(10), 2131-2145. https://doi.org/10.1038/s41418-019-0365-4.