The methods for gene knockout in cells include gRNA plasmid-based, lentivirus-based, and RNP-based methods. The core of the RNP method lies in the assembly of Cas9 protein and sgRNA into an RNP (ribonucleoprotein) complex in vitro. This complex is then introduced into the target cells through electroporation. Gene knockout is achieved through homology-directed repair (HDR) or non-homologous end joining (NHEJ). The lentivirus-based method involves delivering the CRISPR/Cas9 gene editing system into the host cell via a lentiviral vector. Lentiviral vectors are ideal tools for gene delivery due to their stable genomic integration and broad cell transfection capabilities, significantly enhancing the gene editing efficiency of target cells.

Conditional gene knockout technology is used to study potentially lethal genes. The Cre-LoxP system is commonly used in conditional knockout. In this approach, LoxP sites are inserted on both sides of the target gene to create a “floxed” gene. Without Cre recombinase, the gene remains active. When Cre recombinase is introduced, it recognizes the LoxP sites and excises the gene sequence between them, resulting in gene knockout.

We can design gene knockout cell line solutions based on customer needs, including small fragment knockout, large fragment knockout, and frameshift mutations. In small fragment knockout, gRNA is designed in the introns flanking the exons. For frameshift knockout, gRNA is placed in the exons. In large fragment knockout, the entire coding sequence of the gene is knocked out to achieve a large fragment knockout.

hepa 1-6 Cell Line T7EI Detection Results

1. How to select monoclonals for gene knockout cells?

Answer: monoclonals can be selected using the limited dilution method by plating single cells on a 96-well plate. After culturing for a period of time, the formation of monoclonals can be observed under a microscope. Once the single cells have amplified to a sufficient number, monoclonal selection and identification can be performed.

2. What are the challenges in constructing gene knockout cell lines?

Answer: Challenges include delivery methods, culturing, transfection, and monoclonal production. It is recommended to use RNP for delivery, as it offers high efficiency and low off-target effects. Optimizing cell culture conditions, transfection methods, and monoclonal production is crucial to ensure the best conditions before performing the knockout experiments.

3. How to determine the transfection efficiency of cells when using the CRISPR-Cas9 system?

Answer: Fluorescent protein reporter genes: Transfect EGFP mRNA or other fluorescent protein mRNAs to determine the transfection efficiency of the cells.