Random Plasmid Integration
Definition
Plasmid transfection is a non-viral gene delivery technique used to introduce plasmid DNA into cells for the analysis of gene function, expression, and cellular responses. Common approaches include chemical transfection reagents and electroporation.
The following sections focus on chemical-mediated plasmid transfection, while electroporation is discussed separately.
Basic principle
Commonly used transfection reagents include Lipofectamine, CaPO₄, FuGene, and PEI. The principles are essentially the same:
1. Plasmid and transfection reagent are mixed in vitro. The plasmid DNA is negatively charged, while the transfection reagent is positively charged. The transfection reagent encapsulates the plasmid DNA to form a stable complex in vitro.
2. The positively charged complex contacts the negatively charged cell membrane, penetrates the cell membrane, and forms an endosome (a membrane-bound organelle in eukaryotic cells, a vesicle-like structure that is a key transport mechanism during endocytosis).
3.The exogenous plasmid DNA is released in the cytoplasm and enters the nucleus through the nuclear pores, contributing to the expression of the exogenous gene.
Transient Expression and Stable Integrated Expression
Common plasmid transfection lacks a specific, site-specific integration mechanism. Therefore, when the plasmid is delivered into the cell nucleus, integration occurs randomly, and only a small portion of the plasmid is repaired by DNA breaks during cellular replication and integrated into the host chromosomal genome.
Thus, we summarize several key characteristics:
1. Random integration leads to low integration efficiency, and integration into active transcriptional regions is not always possible;
2. A large amount of unintegrated plasmid (for example, using 1x10e6 transfection of 5 μg, for a pcDNA3.1 vector plus a 3000bp target gene,
approximately 5.37*10e11 (The number of copies is gradually diluted or degraded as cells divide.)
3. A peak of transient expression typically occurs within 24-96 hours.
4. The integrated copies must undergo antibiotic screening to obtain positive cells, and resistance must be maintained later.
5. Because the integration occurs through DNA break repair and recombination, there is a possibility of further breaks at the same site later,
potentially disrupting the target gene. Therefore, stability is a challenge. It is recommended to establish a monoclonal stable cell line and analyze its stability through passage.
6. Linear DNA integrates more efficiently than circular DNA, but it is more difficult to deliver than circular DNA.
7. Different cell types vary significantly, manifesting in both delivery and integration.
8. Delivery chemicals are somewhat toxic, and a balance must be struck between delivery efficiency and toxicity.
9. The delivered plasmid must be free of endotoxins, as endotoxins can trigger an immune response.
Application scenarios
Transient transfection
seeking transient high-copy/supercopy protein expression
Stable cell line construction
with a low biosafety level and less stringent requirements for integration efficiency and site
Stable cell line construction service
Reqbio provides stable cell line development services based on the "Plasmid Random Integration System." To date, we have successfully generated over 500 stable cell line models, demonstrating extensive experience in site-specific genome engineering. Please contact us to discuss your project requirements.
Other stable cell lines
Lentiviral Integration
Plasmid Electroporation
Flp-FRT Targeted Integration
Transposon-Based Integration
If you are interested in ordering, please contact us.
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