Knockout cell lines are cell lines obtained by knocking out (i.e. deleting or inactivating) specific genes through genetic engineering technology. Gene knockout can be achieved through a variety of methods, such as ZFN, TALENs, CRISPR, etc. By knocking out specific genes, researchers can study the function of the gene in cellular physiological and pathological processes. In modern biomedical research, gene knockout (KO) cell lines have become an indispensable tool. They not only play an important role in basic research, but also have a wide range of applications in drug development and disease model construction.

Application of KO cell lines

1. Basic research

KO cell lines are widely used in basic research to study gene function. By comparing the phenotypic differences between wild-type cells and KO cells, researchers can reveal the role of specific genes in cell growth, differentiation, signal transduction and other processes. For example, by knocking out the p53 gene, researchers discovered its key role in cell cycle regulation and tumor suppression. By comparing the growth curves, cell cycle distribution and apoptosis rates of p53 KO cells and wild-type cells, the function of p53 in cell stress response was revealed.

2. Disease model

KO cell lines can be used to construct disease models. By knocking out genes related to a certain disease, a cell model of the disease can be simulated, thereby providing a platform for disease mechanism research and drug screening. For example, researchers constructed a cell model of Alzheimer's disease by knocking out the APP gene. By comparing the accumulation of β-amyloid protein in APP KO cells and wild-type cells, the role of APP in the pathogenesis of Alzheimer's disease was revealed.

3. Drug development

During drug development, KO cell lines can be used to verify the effectiveness and specificity of drug targets. By knocking out drug target genes, the effects of drugs on cells can be evaluated, thereby screening out more effective and safe drugs. For example, by knocking out the EGFR gene, researchers compared the drug sensitivity of EGFR KO cells and wild-type cells, verifying the effectiveness of EGFR as an anticancer drug target.

4. Gene function verification

KO cell lines can also be used to verify the effectiveness of gene editing tools. Knocking out specific genes using CRISPR-Cas9 technology can verify the editing efficiency and specificity of the technology. For example, researchers successfully applied the CRISPR nuclease spCas9 from Streptococcus pyogenes to mammalian cell gene editing, demonstrating the ease of programmability and wide applicability of RNA-guided nuclease technology.

Guide to KO cell line construction

Currently, the CRISPR/Cas9 system is widely used. Its principle is to use gRNA to specifically recognize the target sequence and guide the Cas9 endonuclease to cut the PAM upstream of the target sequence, causing a double-strand break in the target site DNA. Then, the organism's DNA damage repair response mechanism is used to connect the sequences at both ends of the break to achieve the knockout of the target gene.

1. Design sgRNA

First, it is necessary to design sgRNA for the target gene. The design of sgRNA needs to consider specificity and effectiveness, and can be designed using online tools.

(1) Select the target gene: determine the gene to be knocked out.

(2) Design sgRNA sequence: use online tools to design sgRNA sequence for the target gene.

(3) Evaluate off-target effects: select sgRNA sequences with low off-target effects to improve editing specificity.

2. Preparation of sgRNA and Cas protein

(1) Synthesize the designed sgRNA.

(2) Prepare Cas protein.

3. Transfect cells

Incubate Ca9 protein and sgRNA to prepare RNP complex. Take an appropriate amount of cells and mix with RNP complex and transfer them to the electroporation cup. Select appropriate electroporation parameters for electroporation. After electroporation, inoculate the cells into the prepared culture medium. Extract the transfected cell genome 48-72 hours after transfection, and perform PCR and sequencing to analyze the gRNA cutting efficiency.

4. Monoclonal screening

Use single cell cloning technology to screen the transfected cells for monoclonal screening. Commonly used methods include limiting dilution and flow cytometry. The selected monoclonal cells need to be genotyped to confirm whether the target gene has been successfully knocked out.

(1) Limited dilution method: dilute the transfected cells and inoculate them into a 96-well plate, one cell per well.

(2) Flow cytometry: Use a flow cytometer to sort single cells and inoculate them into a 96-well plate.

(3) Cultivate monoclonal cells: Cultivate monoclonal cells to a sufficient number.

(4) Genotype identification: Verify whether the target gene is successfully knocked out through PCR and sequencing.

5. Phenotypic analysis

Finally, perform phenotypic analysis on the obtained KO cell line. The knockout effect of the target gene can be verified by Western blot, qPCR, immunofluorescence and other methods, and related functional studies can be conducted.

(1) Western blot: Detect the expression level of the target protein to verify the gene knockout effect.

(2) qPCR: Detect the mRNA expression level of the target gene to verify the gene knockout effect.

(3) Immunofluorescence: Observe the localization and expression of the target protein in the cell.

(4) Other types of functional studies: According to the research purpose, functional experiments such as cell proliferation, apoptosis, and migration are conducted.

Advantages and challenges of KO cells

KO cell lines, as a powerful research tool, have broad application prospects in biomedical research. However, the construction process of KO cell lines is relatively cumbersome, the experimental cycle is long, and it is easy to have incomplete knockout effects, which hinders research progress.

1. Advantages

• High efficiency: Modern gene editing technology makes gene knockout more efficient and convenient.
• Specificity: By designing specific gene editing tools, precise knockout of target genes can be achieved.
• Versatility: Gene knockout cells can be used not only for basic research, but also for disease model construction and drug development.

2. Challenges

• Off-target effects: Although modern gene editing technology has high specificity, off-target effects may still exist, resulting in the editing of non-target genes.
• Cell adaptability: Different types of cells have different adaptability to gene editing tools, and transfection conditions may need to be optimized.
• Phenotypic complexity: Gene knockout may lead to complex phenotypic changes, requiring comprehensive phenotypic analysis.

Knockout Cell Line Generation Services at Creative Biogene

Creative Biogene offers comprehensive editing services, including single-gene knockouts, multi-gene knockouts, and targeted fragment deletions. Our end-to-end pipeline encompasses gRNA design, Cas9 delivery, positive clone screening, genotype verification, and functional validation, achieving an optimal balance between editing efficiency and long-term stability to ensure high success rates, consistent outcomes, and accelerated project timelines.