Cell Line Information
● Cell line name: HL60 (human promyelocytic leukemia cells)
● Gene name: atp6v1a
● Cell type: Polyclonal
● Species: Human
● Cell morphology: Suspended cells, promyelocytes
● Complete culture medium composition: IMDM + 20% FBS + 1% PS
● 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 (the transcriptional repression domain of the KRAB‑1 protein)—binds to the TRE promoter, thereby suppressing shRNA transcription and leaving the target gene’s expression unaffected. Upon addition of tetracycline or other analogs (e.g., doxycycline), 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 atp6v1a 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, investigations 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

Subculture of suspension cells can be performed by either replenishing with fresh medium or replacing the existing medium; the specific procedure is as follows:
1) A small aliquot of the cell suspension was removed for cell counting and viability assessment; when the cell density reached 1.5 × 10^6 cells/mL, subculturing was performed.
2) Add an adequate number of cells to a culture flask containing fresh culture medium, maintaining the cell density at 7 × 10^5 cells/mL.
3) Place the culture flask upright in a 5% CO2, 37°C incubator for cultivation; if the cells reach the density required for passaging, proceed with subculturing.
4) Media change: Every 2–3 days. Note: Use culture flasks with filter‑cap lids to maintain the appropriate levels of air and CO₂ in the medium.

● Cell Cryopreservation

After centrifugation, add an appropriate volume of cryopreservation solution (to achieve a cell density of 10^6 cells per tube), place the cryovials in a cryobox, and store them at −80°C. After 24 hours, transfer the vials to liquid nitrogen for long-term storage.

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