Cell Line Information
● Cell type: iPSC (induced pluripotent stem cell)
● Mutant gene: lrrk2
● Mutation site: G2019S (Gly→Ser, GGC→AGC)
● Species: Human
● Cell morphology: adherent, epithelial-like
● Complete medium composition: specialized medium
● 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 ： After a double-strand break is induced in genomic DNA, an additional homologous repair template must be provided to the cell to enable repair via homologous recombination and to introduce a mutation at the target site. This homologous template sequence is delivered into the cell 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 were introduced, thereby minimizing any off‑target effects on non‑edited genomic loci. Single‑clone cells were isolated from the edited cell pool and subjected to deep sequencing to obtain the expected point‑mutant clones.

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

● Validation data:

Cell Culture Instructions

● Cell recovery

1) Coat the cell culture dish or plate with Matrigel for 30–60 minutes. It is generally recommended to resuscitate the cells in a T25 flask.

2) Remove the cryopreserved cells and immediately place them in a 37°C water bath for rapid thawing. Once the cell suspension in the cryovial has completely melted, promptly transfer it to a centrifuge tube containing 5 mL of cell culture medium, and centrifuge to pellet the cells (refer to the following centrifugation conditions: 1000 rpm, 5 min).

3) Carefully remove the supernatant. Add 1 mL of fresh mTESR cell culture medium, gently mix, and transfer the cell suspension to the pre-coated culture vessel. On the day of cell seeding, add 10 µM Y27632 to promote cell adhesion.

4) On the second day, replace the culture medium with Y27632‑free medium; thereafter, perform daily medium changes using standard culture medium.

● Cell passage

Adherent cells can be passaged when they reach 70%–80% confluence, to prevent overcrowding from compromising cell health. The typical split ratio is 1:10; however, the exact ratio may be adjusted by the experimenter based on the desired growth rate of the cells.

1) Prior to cell passage, the cell culture vessels must be pre-coated with Matrigel.

2) Rinse the cells once with Accutase digestion solution, then add Accutase and incubate in a 37°C incubator for 5 minutes, until the cells become loose and flow‑like. Transfer the cells to a 15 mL centrifuge tube, and add 7–10 mL of standard serum‑free culture medium to neutralize the digestion solution.

3) Place in a centrifuge and centrifuge at 1000 rpm/300 g for 5 minutes.

4) Remove the supernatant and resuspend the cells in 1 mL of culture medium. Then, seed the cells into pre-coated wells.

● Cell cryopreservation

Following the cell passage procedure described above, digest and centrifuge the cells. If the cells are to be passaged again, prepare pre-coated microtiter plates in advance; if they are to be cryopreserved directly, no pre-coated plates are required.

1) After centrifuging the cells, discard the supernatant and resuspend the cells in a rapid cell cryopreservation solution (serum-free). Transfer the cells to cryovials.

2) For one bottle of T25 cells at approximately 70% confluency, use 2 mL of cryopreservation solution, aliquoting 1 mL per vial.

Click the link below to view detailed information.

Product Manual | Grainman 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]