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EDITGENE CO., LTD
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FAQ
How to choose the appropriate gene delivery method?
Selecting a suitable gene delivery system requires a comprehensive assessment based on specific experimental conditions, research objectives, and cell types. Quantitatively comparing various systems in terms of delivery efficiency, cytotoxicity, and stability is an important step in determining the choice.
Viral delivery systems are suitable for experiments that require high delivery efficiency and sustained gene expression, especially when cells can tolerate higher levels of cytotoxicity and immune responses. If lower cytotoxicity and immune response, along with ease of use and cost-effectiveness, are priorities, then a liposome-based gene delivery system should be chosen. For high delivery efficiency that involves delivering large DNA fragments, and if the user can accept a higher operational complexity, a gene gun delivery system is an optional method. If high delivery efficiency is needed while maintaining relative simplicity and no special equipment is required, then the electroporation delivery system may be a suitable choice.
Viral delivery systems are suitable for experiments that require high delivery efficiency and sustained gene expression, especially when cells can tolerate higher levels of cytotoxicity and immune responses. If lower cytotoxicity and immune response, along with ease of use and cost-effectiveness, are priorities, then a liposome-based gene delivery system should be chosen. For high delivery efficiency that involves delivering large DNA fragments, and if the user can accept a higher operational complexity, a gene gun delivery system is an optional method. If high delivery efficiency is needed while maintaining relative simplicity and no special equipment is required, then the electroporation delivery system may be a suitable choice.
How to choose the appropriate vector type?
When selecting a vector, consider the purpose of the experiment and the type of host cells. For example, plasmid vectors are commonly used for gene expression or amplification in bacteria, while viral vectors are more suitable for gene transfer in mammalian cells. Additionally, the vector's promoter, replicon, and antibiotic selection markers should be chosen based on specific requirements.
What host bacteria are used for vector construction in EDITGENE? What type of strains can customers use to amplify plasmid vectors?
During vector amplification, Escherichia coli (E. coli) strains are typically used. The commonly used strain for most non-recombinant vectors is DH5α, which is suitable for most applications. For recombinant vectors, such as lentiviral vectors and transposon vectors, the Stbl3 strain can be used for amplification. Stbl3 is a specialized E. coli strain derived from HB101, which has a mutation in the recombinase gene recA13, effectively suppressing recombination of long fragment terminal repeat regions and reducing the likelihood of erroneous recombination.
What host bacteria are used for vector construction in EDITGENE? What type of strains can customers use to amplify plasmid vectors?
During vector amplification, Escherichia coli (E. coli) strains are typically used. The commonly used strain for most non-recombinant vectors is DH5α, which is suitable for most applications. For recombinant vectors, such as lentiviral vectors and transposon vectors, the Stbl3 strain can be used for amplification. Stbl3 is a specialized E. coli strain derived from HB101, which has a mutation in the recombinase gene recA13, effectively suppressing recombination of long fragment terminal repeat regions and reducing the likelihood of erroneous recombination.
What is lentivirus packaging?
Lentivirus is a gene delivery tool that introduces exogenous genes into cells. By using tool cells such as 293T, lentiviral vectors carrying target DNA fragments are packaged into lentiviral particles with cell-infectious activity. This packaging process includes constructing lentiviral vectors, preparing packaging plasmids, culturing tool cells, transfecting plasmids, collecting viral particles, purifying and concentrating viral particles, and titration.
Why choose lentivirus as a gene delivery tool?
Lentiviruses have high transduction efficiency and the ability to maintain stable gene expression over the long term, making them particularly suitable for cell types that are difficult to transfect. Furthermore, lentiviruses can integrate exogenous genes into the host genome, ensuring prolonged gene expression.
After lentiviral transduction, the cell condition is poor, and there is significant cell death. How can this be addressed?
4.Poor cell condition after lentiviral infection may be caused by various factors. Here are some possible reasons and corresponding solutions:
1.High viral titer: High titers of lentivirus may cause cytotoxicity, preventing normal cell growth. Solution: Lower the viral titer and conduct a series of dilution experiments to find a titer that effectively transduces without adversely affecting cell growth.
2.Poor cell condition: The health status of cells before infection can affect growth after infection. Solution: Ensure cells are in optimal condition for infection, for example, by changing to fresh culture medium 24 hours before infection and ensuring appropriate cell density.
3.Toxicity of gene expression mediated by the virus: The gene carried by the lentiviral vector may be toxic to the cells, affecting their growth. Solution: If possible, use a control vector to determine if the problem is related to gene expression, and select appropriate vectors or genes for research.
4.Excessive antibiotic selection pressure: If antibiotics are used to select transfected cells, excessive concentrations of antibiotics may inhibit cell growth. Solution: Optimize the antibiotic concentration and use gradient experiments to determine the optimal concentration.
What is Prime Editing?
Prime Editing is a novel gene editing technology that enables precise gene editing without introducing double-strand DNA breaks. It has two core components: pegRNA and the PEmax gene-editing enzyme (Cas9n-RT). PegRNA not only targets the desired sequence but also contains the base modification information. In the editing system, pegRNA guides PEmax to the designated edit site, nicks the DNA single strand, and reverse transcribes the sequence within the pegRNA to modify, inserting it into the target genome location, thereby achieving precise single-base substitutions or small insertions and deletions
Why choose EDITGENE’s point mutation services?
EDITGENE’s newly upgraded seventh-generation Bingo™ Prime Editing (PE7) platform optimizes editing protein and RNA editing activity. Compared to the fifth-generation PE technology, point mutation success rates and gene editing efficiency have significantly improved, with one-on-one support from PhDs from globally renowned institutions.
How does EDITGENE’s Bingo™ Prime Editing 7 (PE7) platform ensure point mutation success rates?
EDITGENE’s Bingo™ Prime Editing 7 (PE7) platform is built upon over ten years of gene editing experience, with optimization and advancements derived from thousands of gene editing CRO projects, achieving significantly higher success rates than traditional site-specific mutation systems. The Bingo™ Prime platform utilizes highly efficient reverse transcriptase and precise guide RNA design, ensuring each point mutation reaches the desired outcome.
Are all types of genes suitable for KO cell lines?
Not all genes are suitable for knockout. Some gene knockouts may result in cell death or severe dysfunction, particularly for essential genes. In such cases, conditional knockouts or gene knockdowns (e.g., RNAi) may be used instead.
What is a KO cell line?
KO (Knockout) cell line is a cell line where a specific gene has been completely removed or rendered non-functional through gene editing technologies such as CRISPR-Cas9. These cell lines are critical for understanding gene functions and disease mechanisms.
What is the difference between KO cell lines and gene knockout animal models?
KO cell lines are used for in vitro experiments, suitable for high-throughput screening and cellular studies, while gene knockout animal models are used for in vivo experiments to study gene functions within an entire organism and its interaction with the environment.
Why do researchers use KO cell lines?
Researchers use KO cell lines to investigate gene functions by observing the effects of gene deletion on cellular behavior. This helps in understanding the role of genes in various processes like cell growth, metabolism, and signal transduction. KO cell lines are vital for studying diseases like cancer, genetic disorders, and neurodegenerative diseases.
Why choose EDITGENE?
EDITGENE provides access to a comprehensive library of over 4,500 high-quality knockout (KO) cell lines, enabling researchers to save valuable time. Our custom gene knockout services are highly efficient, boasting a high positive rate while minimizing off-target effects. Clients also benefit from personalized, one-on-one support from a team of PhD experts from globally renowned institutions, ensuring top-tier service and results.
