KC-6402

293T-TOP1-R364H-KI Cell Line

63973

Home » 293T-TOP1-R364H-KI Cell Line

Background of 293T-TOP1-R364H-KI Cell Line

DNA topoisomerase I (TOP1) is an essential nuclear enzyme that relaxes supercoiled DNA during transcription and replication by catalyzing transient single-strand breaks. This enzyme is encoded by a single-copy gene located on human chromosome 20q12. TOP1 activity is critical for normal cellular processes, including embryonic development and immune function. Notably, TOP1 inhibitors, such as camptothecin and its derivatives topotecan and irinotecan, are among the most effective anticancer drugs used clinically. These agents function as TOP1 poisons by stabilizing the TOP1-DNA cleavage complex (TOP1cc), converting it into a lethal DNA lesion that triggers cell death. However, cancer cells often develop resistance through mechanisms involving TOP1 downregulation, enhanced repair pathways (e.g., TDP1 activation), or altered DNA damage responses. Recent therapeutic advances include antibody-drug conjugates (ADCs) that deliver TOP1 inhibitors specifically to tumor tissues, as well as combination strategies targeting both TOP1 and DNA damage response pathways to overcome resistance.

Specifications

Catalog Number	KC-6402
Cell Line Name	293T-TOP1-R364H-KI Cell Line
Clone Number	1A3
Host Cell Line	293T
Description	Stable 293T clone expressing endogenous TOP1 gene bearing R364H mutations
Quantity	One vial 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
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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-TOP1-R364H-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-TOP1-R364H-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-TOP1-R364H-KI cell line stable clone using RT-PCR sequencing.

Figure 3. Characterization of dose-response curves for TOP1 inhibitors on 293T and 293T-TOP1-R364H-KI cells.

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 1:3-1:6 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

Venkatachalam A, Kaufmann SH. Targeting DNA Topoisomerase I for the Treatment of Cancer: Past, Present and Future. Journal of Molecular Biology. 2025;169401.
Majumdar AG, Chauhan N, Gupta P, et al. CHK1-mediated regulation of TOP1 catalytic activity suppresses replication and transcription-associated genomic instability. 2024.
Pommier Y. DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition. Chemical Reviews. 2009;109(7):2894-2902.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.