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.
