Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase

Abstract

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

Active Nuclear Import of Mammalian Cell-Expressible DNA Origami

“…Nuclear import remains a major barrier to successful gene delivery due to the impermeability of the nuclear membrane . It is predominantly hypothesized that delivered genetic material passively enters the nucleus during mitosis when the nuclear envelope breaks down. , However, this passive entry is relatively inefficient, and only a small amount of the delivered DNA reaches the nucleus . Furthermore, most cells in vivo are either postmitotic (nondividing) or have very long doubling times, limiting entry via passive nuclear transport during mitosis. , …”

Enhanced Gene Delivery Triggered by Dual pH/Redox Responsive Host‐Guest Dimerization of Cyclooligosaccharide Star Polycations

“…In the case of SS-bisAd, however, the high concentration of glutathione (GSH) in the cytosol would promote disulfide bridge cleavage, thereby deactivating the vector for strong pDNA recapture. This is critical for enabling subsequent nuclear entry of pDNA: although some examples have demonstrated the presence of cationic polymer molecules in the nucleus during polyplex-mediated transfection, [21] the current evidence supports that pDNA must be essentially free or loosely bound to undergo either integration into the nucleus during nuclear envelop reformation at telophase in dividing cells [22] or, eventually, active import through the nuclear pore complex [23] (Figure 4). In CT2 derivatives the cavity is collapsed; [24] dissimilarities in pDNA complexation and transfecting properties between 1 and 2 in combination with the ditopic adamantane derivatives should then be principally ascribed to the host-guest component of the process.…”

Genetically Encoded DNA Origami for Gene Therapy In Vivo

“…Furthermore, after delivery by lipo 2000 transfection or electroporation, the G EGFP -DO both demonstrated a noticeably high EGFP expression (Figure S4B). Meanwhile, as shown in Figure S4C, G EGFP -DO also elicited a higher EGFP expression in the dividing cells than in the arrested cells by treatment with aphidicolin (a blocker for cell division), which is attributed to the passive nuclear import by nuclear envelope reformation during mitosis . These results indicate that genetically encoded DNA origami can be successfully employed as the gene carrier for gene delivery.…”

“…Meanwhile, as shown in Figure S4C, G EGFP -DO also elicited a higher EGFP expression in the dividing cells than in the arrested cells by treatment with aphidicolin (a blocker for cell division), which is attributed to the passive nuclear import by nuclear envelope reformation during mitosis. 63 These results indicate that genetically encoded DNA origami can be successfully employed as the gene carrier for gene delivery. For cellular uptake, the genetically encoded DNA origami was employed as the template for lipid growth.…”