KC-4409

T47D-RB1-KO-1A1-Cell-Line

46096

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Background of T47D-RB1-KO-1A1-Cell-Line

The Retinoblastoma 1 (RB1) gene, located on chromosome 13q14, is a tumor suppressor gene essential for cell cycle regulation. The protein encoded by this gene is a negative regulator of the cell cycle and was the first tumor suppressor gene found. The encoded protein also stabilizes constitutive heterochromatin to maintain the overall chromatin structure. The active, hypophosphorylated form of the protein binds transcription factor E2F1. Defects in this gene are a cause of childhood cancer retinoblastoma (RB), bladder cancer, and osteogenic sarcoma.

Specifications

Catalog Number	KC-4409
Cell Line Name	T47D-RB1-KO-1A1-Cell-Line
Host Cell Line	T47D
Description	Stable T47D clone with human RB1 gene knockout, No.1A1
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS+0.2 Units/mL Insulin
Selection Marker	N/A
Morphology	epithelial
Subculture	Split saturated culture 1:3-1:5 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 60 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

T47D-RB1-KO-1A1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of T47D-RB1-KO-1A1 cell line stable clone using PCR sequencing.

Figure 2: Characterization of T47D-RB1-KO-1A1 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of Dose-response curves and IC50 values for T47D and T47D-RB1-KO-1A1 cells treated with Palbociclib, Abemaciclib, and Cisplatin over 5 days.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS+0.2 Units/mL Insulin)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 1:3-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Classon M, Harlow E. The retinoblastoma tumour suppressor in development and cancer. Nat Rev Cancer. 2002 Dec;2(12):910-7. doi: 10.1038/nrc950. PMID: 12459729. 2.Knudsen ES, Wang JY. Targeting the RB-pathway in cancer therapy. Clin Cancer Res. 2010 Feb 15;16(4):1094-9. doi: 10.1158/1078-0432.CCR-09-0787. Epub 2010 Feb 9. PMID: 20145169; PMCID: PMC2822892. 3.https://www.ncbi.nlm.nih.gov/gene/5925