Point mutation refers to the introduction of a single nucleotide mutation at a specific site using CRISPR/Cas9 or other gene editing technologies, including base substitutions, insertions, or deletions. Point mutation cells can be used in gene function research, disease mechanism research, drug development, and evolutionary studies.

 

See the LiMan official account: Technical Topic | Common Application Scenarios of Gene Point Mutation Cell Lines [Collection]

 

However, successfully constructing a point mutation cell line is not simple. LiMan Biology provides one-on-one customized services, designing the most suitable technical solutions for customer needs, making your scientific research more efficient.

 

LiMan provides in vitro gene point mutation cell line construction services for eukaryotic cells. For this service, the LiMan team will regularly update Q&A to better serve our customers. Q&A [2411 version] is as follows:

 

1. What is the principle of the method used by LiMan's point mutation service?

 

The theoretical basis for constructing a cell point mutation model is homologous recombination repair. When double-stranded genomic DNA is broken due to external factors, the existing DNA repair mechanisms in the cell will be activated to repair the broken DNA double strand. Depending on the repair method, it can be divided into homologous recombination repair (HR) and non-homologous recombination repair (NHEJ). Homologous recombination repair (HR) refers to the perfect repair of DNA using homologous DNA sequences as a template during repair. Non-homologous end joining (NHEJ) refers to the direct reconnection of the broken ends of DNA by related proteins in the cell, which may randomly introduce or delete a certain number of bases. By using appropriate methods to promote the homologous recombination repair of cells while introducing donor DNA containing specified mutations, point editing of the target DNA can be accomplished.

 

2. What information do I need to provide when consulting about point mutation needs?

You only need to provide the cell name, target Gene Symbol, and point mutation site information (for example: A549 cell point mutation of the 42nd amino acid of gene A, AAG mutated to AGG)

 

3. What types of point mutations do you perform?

Generally, all mutation types can be performed. Theoretically, mutations within 30 bases belong to point mutations, including but not limited to missense mutations, synonymous mutations, and nonsense mutations. Specific requirements depend on the customer's needs.

 

4. What is the success rate of point mutation?

The success rate of point mutation is related to the cell and the target. When repairing double-stranded DNA, the probability of homologous recombination is between 0.3% and 5%. LiMan Biology has developed a unique method that can significantly increase the probability of homologous recombination repair (8-12%) in most cell lines. Combined with our automated monoclonal separation and sequencing analysis, the success rate of isolating monoclonal cells can be effectively improved.

 

5. What are the advantages of your point mutation services?

① Technical Advantages ：For the point mutation cells constructed using the above method, no additional reporter or resistance genes are introduced, which can minimize the impact on non-edited sites in the genome.

② Scheme Design ：More than 10 years of overseas research team specializing in gene editing, carefully optimizing the design of point mutation schemes, comprehensively considering the PAM region of sgRNA cleavage, cleavage specificity, the position and length of the donor sequence and modification, and the binding site of sequencing verification primers and other details.

③ Transfection Advantages ：LiMan Biology has successfully edited more than 3000 cells, guaranteeing transfection efficiency and significantly improving the success rate of point mutations.

④ Monoclonal Screening ：LiMan Biology has achieved automated monoclonal plating and selection. Through efficient monoclonal sequencing, the editing success rate is greatly improved.

6. How long is the cycle for point mutation?

The cycle of cell line development is related to the cell doubling time. The shorter the doubling time, the faster the development cycle. For example, if the cell doubling time is within 24 hours, the cycle for obtaining a point mutation cell line is 8-10 weeks, and the cycle will be adjusted according to the cell doubling time.

 

7. What are the deliverables of the point mutation service?

① Cell Line ：Point mutation positive monoclonal cell line. Our company provides various cell delivery methods, such as simultaneous delivery of WT cells and point mutation cells, and customers can choose cryopreservation tubes or T25 live cells.

② Point mutation cell line construction report ：Detailed description of cell culture methods, cell construction process, Sanger sequencing report and original data. Our company provides all after-sales services related to cell lines, including ensuring that cells are free of contamination, stable passage of genotypes, ensuring that customers can successfully revive cells, and providing services such as Sanger verification data analysis after multiple passages.