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EDITGENE CO., LTD
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FAQ
Does the piggyBac Transposon System Kit recommend lipofection or electroporation?
The PB system in this kit includes plasmids compatible with lipofection, electroporation, or other transfection methods. You can select the transfection method and parameters based on cell type. Most adherent cells are compatible with various transfection methods, while electroporation is recommended for therapeutically relevant cells.
What should I do if the transfection efficiency in target cells is low?
Before the main experiment, conduct a transfection pre-experiment to explore different transfection methods and optimize conditions. Efficient transfection is essential for successful gene transposition.
How can I verify the transposition activity of the piggyBac Transposon System Kit?
The transposon plasmid in this kit contains the CopGFP gene. Successfully transposed cells will exhibit green fluorescence under a fluorescence microscope. Due to cell heterogeneity, transfection and transposition efficiencies may vary across cell types. To minimize transposition variability, optimize transfection methods for target cells.
Can cells constructed with the piggyBac Transposon System Kit be stably passaged?
Yes. The piggyBac Transposon System Kit targets genomic DNA for gene transposition, allowing the genotype of target cells to be stably inherited.
Note: Maintain transposed cells with half-dose puromycin for stability.
Does the Bingo™ CRISPR Point Mutation Cell Line Generation Kit recommend lipofection or electroporation?
The Bingo™ PE, pegRNA, and gRNA in the kit are all plasmids, compatible with both lipofection and electroporation. You can choose the appropriate transfection method and parameters based on cell type. Generally, adherent cells can be transfected by either method, while therapeutically relevant cells are recommended to use electroporation
Why do cells plated in a 96-well plate for single-clone screening grow slowly or die, even though single-clone formation was good in the pre-experiment?
Gene mutations may affect cell viability. Before conducting a gene point mutation experiment, it is advisable to consult relevant literature to understand the function of the target gene. If the target gene plays a critical role in cell proliferation or survival, point mutations may hinder these processes, making it difficult to obtain positive cells.
How can I verify the editing activity of the Bingo™ CRISPR Point Mutation Cell Line Generation Kit?
The kit’s Positive Control has been validated in HeLa cells (human gene) and N2a cells (mouse gene). Due to the high heterogeneity of cells, transfection and editing efficiency may vary in different cell types using the same kit.
Can cells constructed with the Bingo™ CRISPR Point Mutation Cell Line Generation Kit be stably passaged?
Yes. The Bingo™ CRISPR Point Mutation Cell Line Generation Kit targets genomic DNA for gene mutation, allowing the genotype of the target cells to be stably inherited by subsequent generations.
What should I do if the transfection efficiency in target cells is low?
It is recommended to conduct a pre-transfection experiment before the main experiment. Try different transfection methods to find optimal conditions, such as common chemical transfection methods (e.g., lipofection) or physical methods (e.g., electroporation).
What are induced pluripotent stem cells (iPSCs)?
Induced pluripotent stem cells (iPSCs) are cells reprogrammed from adult cells to a pluripotent state. They exhibit similar characteristics to embryonic stem cells, capable of differentiating into nearly all cell types in the body. This technology allows scientists to generate various cell types in vitro for research and therapy without the need for embryonic stem cells.
How do iPSCs help us understand complex diseases?
Cells from patients are isolated and reprogrammed into iPSCs, which are then induced to differentiate into specific cell types to create disease models. These models enable researchers to study disease mechanisms, uncover disease-related genes, and molecular pathways, thereby advancing the development of new therapies. By analyzing these cells, scientists can observe disease-related changes at the cellular level, providing new perspectives in disease research.
What is the difference between iPSCs and embryonic stem cells (ESCs)?
Both iPSCs and embryonic stem cells (ESCs) are pluripotent, but iPSCs are derived from reprogrammed somatic cells, while ESCs originate from early embryos. iPSCs do not involve embryo use, making them a more ethically acceptable choice, and they also avoid immune rejection issues, as they can be generated based on a patient’s genetic background.
What are the potential applications of iPSCs in clinical practice?
iPSCs have broad clinical potential, including applications in cell therapy (e.g., for diabetes or heart disease treatment), tissue engineering (e.g., development of artificial skin or liver tissue), and personalized drug screening (e.g., selecting optimal treatments based on a patient’s specific cellular response). These applications may transform treatment methods, offering more effective and personalized medical services.
What are the differences between Cas9, Cas12, and Cas13?
The main differences among Cas9, Cas12, and Cas13 lie in their action mechanisms:
· Cas12 is activated to cleave ssDNA trans-cleaving after binding with guide RNA and target DNA.
· Cas13 is activated to cleave ssRNA trans-cleaving after binding with guide RNA and target RNA.
· Cas9 has not been reported to exhibit trans-cleaving activity.
· Cas12 is activated to cleave ssDNA trans-cleaving after binding with guide RNA and target DNA.
· Cas13 is activated to cleave ssRNA trans-cleaving after binding with guide RNA and target RNA.
· Cas9 has not been reported to exhibit trans-cleaving activity.
What role does gene knock-in play in drug development?
Gene knock-in plays a crucial role in drug development. It is used in target validation by introducing specific genes into cell lines or animal models to confirm drug target efficacy. It also aids in establishing disease models, testing drug efficacy and safety in these models, and supporting drug screening through high-throughput screening in knock-in cell lines to identify potential drug candidates. Additionally, gene knock-in helps uncover drug mechanisms, optimize drug structure, and improve dosing strategies, expediting drug development while enhancing efficacy and safety.
