Cell Line Information
● Cell line name: MCF-7 (human breast cancer cells)
● Gene name: RPN2
● Cell type: Polyclonal
● Species: Human
● Cell morphology: adherent growth, epithelial-like
● Complete medium composition: MEM + 10% FBS + 1% P/S + 10 µg/mL Insulin
● Passage ratio: 1:2–1:4
● Passage frequency: Passaged once every 2–3 days.
● Mycoplasma test: Negative

Gene Editing Information

● Construction Method

Gene knockdown refers to the reduction of the expression level of a target gene in cells or organisms, thereby inhibiting its function, typically achieved through RNA interference (RNAi). RNAi is usually mediated by synthetic small interfering RNAs (siRNAs) or stable hairpin RNAs (shRNAs) that direct the targeted degradation of mRNA. Because RNAi generally results in partial suppression of the target gene’s expression, this approach is termed “gene knockdown.” Gene knockdown enables researchers to investigate the biological functions of specific genes, metabolic pathways, and other aspects, making it a widely used method for studying gene function.

The Tet-On inducible gene expression system enables regulation of the expression level of a target gene in mammalian cells by inducing the expression of an shRNA. Mechanism of the Tet-On–inducible shRNA expression vector: In the absence of tetracycline and its analogs (such as doxycycline), the fusion protein tTS—derived from TeR (tetracycline repressor) and KRAB‑AB (a transcriptional repression domain of the Ki‑1 protein)—binds to the TRE promoter, thereby suppressing shRNA transcription and leaving the expression of the target gene unaffected. Upon addition of tetracycline or other analogs (e.g., doxycycline) to the system, the transcriptional repressor is inactivated, activating shRNA transcription and triggering degradation of the target gene’s mRNA, thus reducing the corresponding protein expression level.

Our company employs a self‑optimized Tet‑On–inducible shRNA knockdown platform to integrate multiple copies of three shRNAs targeting the RPN2 gene into transcriptionally active genomic loci. Following antibiotic selection, we obtain stable cell pools, which are then expanded via limiting dilution to generate stable monoclonal cell lines.

● Application Scenarios

The Tet‑On–based inducible shRNA knockdown system enables temporally controlled, dose‑dependent, and reversible gene silencing through exogenous small‑molecule regulation, and has been widely adopted in cell biology, tumor‑mechanism research, and drug development. It is particularly well suited for: studies of essential gene functions, analysis of time‑dependent signaling pathways, investigation of acute injury models, validation of drug combination mechanisms, and evaluation of molecular glue/PROTAC activities.

● Data Validation

Western blot assay results

Cell Culture Instructions

● Cell Revival

1) Remove the cryovial from the liquid nitrogen tank or the −80°C freezer, and promptly place it in a 37°C water bath, gently swirling it to facilitate rapid thawing. To minimize the risk of contamination, you may first transfer the cryovial into a clean, disposable PE glove before immersing it in the water bath.
2) Once the contents of the cryovial have thawed, remove it immediately and wipe the exterior of the cryovial with a 75% ethanol‑soaked gauze.
3) Place the cryovial in a biosafety cabinet, transfer the contents of the cryovial into a centrifuge tube containing approximately 9 mL of complete culture medium, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
4) Resuspend the cells in an appropriate volume of complete culture medium, transfer them to a new culture flask, and incubate them in a 5% CO2, 37°C incubator under static conditions.

● Cell passage

When cell density reaches 80% or higher, passaging can be performed at a ratio of 1:2 to 1:4, with passages carried out every 2–3 days. The specific procedure is as follows:
1) Aspirate and discard the culture medium from the flask, add an appropriate volume of PBS, gently swirl to rinse the cells, and then aspirate the PBS.
2) Add approximately 1.0 mL of 0.25% trypsin, gently rock the dish to ensure even coverage of the cells, and then incubate in the cell culture incubator for 2–3 minutes. Under the microscope, observe until the cells become rounded; when about 80% of the cells have detached from the surface, add approximately 5 mL of complete culture medium to neutralize the trypsin. Gently pipette up and down to detach the cells from the flask surface, yielding a single-cell suspension. (The volumes specified here are for a T75 flask; adjust accordingly based on your specific experimental conditions.)
3) Transfer the digested cell suspension to a clean 15 mL centrifuge tube, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
4) Resuspend the cells in an appropriate volume of complete culture medium, transfer them to new culture flasks at the recommended passage ratio (1:2 to 1:4), and incubate the cells in a 5% CO₂–37°C incubator under static conditions.

● Cell Cryopreservation

Cell cryopreservation can be performed when cell confluence reaches 80%–90%. The homemade cryopreservation solution consists of DMEM complete medium, 30% FBS, and 10% DMSO. To ensure high viability of the cryopreserved cells, it is best to prepare the solution in advance and pre‑cool it at 4°C.
 

Click the link below to view detailed information.

   Service Guide | Construction of Tet‑on Inducible Knockdown Cell Lines: A Comprehensive Analysis of Frequently Asked Questions and Strategy Selection

[Technical Spotlight] A “Dynamic Switch” for Gene Function Studies: Tet-On–Based Knockdown Solutions and Case Studies!