KC-5872

293T-GSPT1-G575N-KI Cell Line

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Background of 293T-GSPT1-G575N-KI Cell Line

GSPT1 (G1 to S phase transition 1) is a crucial gene encoding eRF3a, a GTPase that partners with eRF1 to form the eukaryotic translation termination complex. The recurrent G575N mutation, a glycine-to-asparagine substitution at codon 575 within the GTP-binding domain, is a significant oncogenic driver in hematological malignancies, particularly acute myeloid leukemia (AML). This mutation profoundly impairs the GTP hydrolysis activity of eRF3a, leading to dysfunctional translation termination. Consequently, it results in widespread ribosome stalling and readthrough of stop codons. This disruption in protein synthesis promotes the stabilization of oncoproteins and genomic instability, thereby fueling leukemogenesis and cancer cell survival. The G575N mutant is clinically associated with aggressive disease progression, poorer overall survival, and resistance to conventional chemotherapy. Its pivotal role in oncogenesis has made it a compelling therapeutic target. Current research is intensely focused on developing targeted agents, such as cereblon E3 ligase modulators (e.g., CC-90009), which selectively degrade GSPT1 protein. These investigational drugs have shown promising pre-clinical efficacy, offering a potential novel strategy for treating AML patients harboring this high-risk mutation.

Specifications

Catalog Number	KC-5872
Cell Line Name	293T-GSPT1-G575N-KI Cell Line
Clone Number	2B3
Host Cell Line	293T
Description	Stable 293T clone expressing endogenous GSPT1 gene bearing G575N mutations
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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	NA
Morphology	Fibroblastoid cells growing as a monolayer
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 28 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-GSPT1-G575N-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-GSPT1-G575N-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-GSPT1-G575N-KI cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of Dose-response curves and IC50 values for 293T and 293T-GSPT1-G575N-KI cells treated with SJ6986 over 5 days.

Figure 4: Characterization of 293T-GSPT1-G575N-KI cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (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 1:4-1:5 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

Parkin B, Londoño-Joshi A, Wang Q, et al. GSPT1 mutations are associated with poor outcome in acute myeloid leukemia. Blood. 2019;134(Supplement 1):138.
Tyner JW, Tognon CE, Bottomly D, Wilmot B. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018 Oct;562(7728):526-531. doi: 10.1038/s41586-018-0623-z. Epub 2018 Oct 17. PMID: 30333627; PMCID: PMC6280667.
Surka C, Jin L, Mbong N, Lu CC, Jang IS, Rychak E, Mendy D. CC-90009, a novel cereblon E3 ligase modulator, targets acute myeloid leukemia blasts and leukemia stem cells. Blood. 2021 Feb 4;137(5):661-677. doi: 10.1182/blood.2020008676. PMID: 33197925; PMCID: PMC8215192.