Cell Line Information
● Cell line name: U251 (human glioma cells)
● Mutant gene: TERT
● Mutation site: -146C＞T (deletion of one C)
● Species: Human
● Cell morphology: adherent, fibroblast-like
● Complete culture medium composition: DMEM + 10% FBS + 1% P/S
● Passage ratio: 1:2 to 1:4
● Passage frequency: 2–3 times per week
● Mycoplasma test: Negative

Gene Editing Information
● Construction method: The theoretical basis for constructing cellular point‑mutation models is homologous recombination repair. When the double strands of genomic DNA are cleaved by external factors, the cell’s DNA repair machinery is activated to repair the broken DNA ends; depending on the repair mechanism, this can be classified as homologous recombination repair (HR) or non‑homologous end joining (NHEJ). Homologous recombination involves using a homologous DNA sequence as a template to achieve precise repair, whereas non‑homologous end joining entails the direct ligation of the broken DNA ends by cellular proteins, which may randomly introduce or delete a variable number of nucleotides. Based on this theoretical framework, the construction of point‑mutation cell lines proceeds in three steps:

1. Site-specific DNA cleavage ： In this process, the Cas9 protein is transfected into the target cells together with the sgRNA. Under the guidance of the sgRNA, the Cas9 protein cleaves the DNA at the target site, generating a double-stranded DNA break.
2. Homologous recombination repair of DNA ： Following double-strand breaks in genomic DNA, to enable repair via homologous recombination and introduce a mutation at the break site, an additional homologous repair template must be provided to the cell. This homologous template sequence is delivered into the cell by transfection together with the sgRNA and Cas9 protein.
3. Positive cell screening ： For the point‑mutant cells generated by the aforementioned method, no additional reporter or resistance genes are introduced, thereby minimizing any off‑target effects on unedited genomic loci. Single‑clone cell lines harboring the intended point mutations are isolated from the edited cell pool and subjected to deep sequencing.

● Application scenarios: Liman Bio’s point‑mutation cell lines precisely mimic single‑base substitutions, making them ideal for elucidating gene function and investigating disease mechanisms. These cells can be used to establish pathological models of tumors, genetic disorders, and other conditions, supporting drug target validation and resistance‑assessment studies, thereby providing a standardized, highly reliable in vitro platform for basic research and new‑drug development.

● Validation data:

Cell Culture Instructions

● Cell recovery

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 liquid in the cryovial has thawed, remove it immediately and wipe the surface 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 the cells 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 a frequency of 2–3 times per week. 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 flask to ensure even coverage of the cells, and then incubate in the cell culture incubator for 2–3 minutes. Under the microscope, observe that the cells have become rounded; when about 80% of the cells have detached from the flask wall, 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, dispersing them into a single-cell suspension. (The volumes specified here are for a T75 flask; adjust the amounts as needed based on your specific experimental conditions.)

3) Transfer the cell suspension after digestion 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 a new culture flask 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 post-thaw viability, it is best 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 flask to ensure even coverage of the cells, and then incubate in the cell culture incubator for 2–3 minutes. Under the microscope, observe that the cells have become rounded; when about 80% of the cells have detached from the flask wall, 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, dispersing them into a single-cell suspension. (The volumes specified here are for a T75 flask; adjust the amounts as needed based on your specific experimental conditions.)

3) Transfer the cell suspension after digestion 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 (recommended: 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 labels. The label should include the cell name, passage number, cryopreservation date, and the operator’s name, among other information.

6) Place the cryovials in a cell‑freezing container, and then transfer the container to a −80°C freezer overnight. After overnight incubation, transfer the vials to a liquid nitrogen tank for long‑term storage.

Click the link below to view detailed information.

Product Manual | LIMAN Easy Point Mutation Cell Line Construction Custom Services and Products

Service Guide | Q&A on LIMAN Point-Mutant Cell Line Construction Services [Version 2411]

Service Guide | Comprehensive Q&A on Site‑Directed Mutagenesis–Based Stable Cell Line Construction: From Concepts to Experimental Details—All in One Article! [Version 2512]