Cell Line Information

● Cell line name: CHO-K1 (hamster ovary cells)

● Gene name: GFP

● Cell type: Polyclonal

● Cell morphology: adherent growth, epithelial‑like cells

● Complete medium composition: DMEM/F12 + 10% FBS + 1% PS

● Passage ratio: 1:2 to 1:4

● Passage frequency: Passaged every 2–3 days.

● Mycoplasma test: Negative

Gene Editing Information

● Construction method

Our company employs a proprietary, optimized random multi-copy transposon‑mediated gene knock-in (Shotgun©) platform to integrate multiple copies of GFP into transcriptionally active genomic loci. Following antibiotic selection, we obtain stable cell pools, which are then expanded via limiting dilution to generate stable, single‑clone cell lines exhibiting high‑copy‑number gene overexpression.

● Application Scenarios

Liman Bio’s overexpression cell lines enable stable, high‑level expression of target genes, supporting gain‑of‑function studies and the elucidation of signaling pathways. They are well suited for disease model development, target validation, and the screening of antibodies and small‑molecule drugs, providing efficient and robust in vitro experimental platforms for basic research and innovative drug discovery.

● Data Validation

GFP overexpression sequence

GFP Gene Overexpression Assay

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 while gently shaking it to achieve 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 liquid in the cryovial has 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 transfection

1) Cell preparation: Perform cell dissociation following the standard subculture protocol. Conduct a cell count, and based on the results, aliquot the appropriate number of cells for subsequent use. Seed at a density of 1.0 × 10^6 cells/mL, with 2 mL per well, in a six-well plate, and incubate at 37°C in 5% CO2 for 24 hours.
2) Prepare the transfection complex (using one well as an example): Add 50 μl of Opti‑MEM to tube 1, along with 2 μg of linearized DNA; add 50 μl of Opti‑MEM to tube 2, along with the transfection reagent. Then, transfer the contents of tube 2 into tube 1, mix thoroughly, and let stand for 10 minutes.
3) Transfection: Add the above complex to a 6-well plate and incubate in a static incubator at 37°C with 5% CO2 for 72 hours.
4) Cellular selection under pressure: Stable cell lines with integrated constructs are selected using puromycin.

● 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 carefully aspirate the PBS.
2) Add approximately 1.0 mL of 0.25% trypsin, gently rock the dish to ensure even coverage, and incubate in the cell culture incubator for 2–3 minutes. Under the microscope, observe until the cells become rounded; then gently tap the side of the flask. 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 confluence of 80%–90% is optimal for cryopreservation. To ensure high viability of the cryopreserved cells, it is recommended to prepare the cryopreservation solution in advance and pre‑cool it at 4°C.
The specific procedures for cell cryopreservation are as follows:
1) Aspirate and discard the culture medium from the flask, then add an appropriate volume of PBS and wash once.
2) Add approximately 1.0 mL of 0.25% trypsin, gently rock the dish to ensure even coverage, and incubate in the cell culture incubator for 2–3 minutes. Under the microscope, observe until the cells become rounded; then gently tap the side of the flask. 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) Add an appropriate volume of pre‑prepared, pre‑cooled cryopreservation solution based on the cell pellet size (we recommend 3 mL of cryopreservation solution per T75 flask), and gently pipette up and down to resuspend the cells.
5) Transfer the cell suspension into cryovials, 1 mL per vial. Label the cryovials in advance or affix pre‑printed labels. The label should include the cell line name, passage number, cryopreservation date, and the operator’s name, among other information.
6) Place the cryovials in a cell‑freezing box, and then transfer the box to a −80°C freezer overnight. After overnight incubation, the vials can be transferred to a liquid nitrogen tank for long‑term storage.

Click the link below to view detailed information.

Service Guide | Q&A on Customized Cell Lines Overexpressing LIM Domain Proteins [Version 2506]

Service Guide | Q&A on the Construction of Stable Cell Lines with Overexpression—From “Is It Feasible?” to “How Can We Do It Reliably?” [Version 2601]