Abstract
The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex- or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07.CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer.
Highlights
The nuclear delivery of plasmid DNA is a crucial rate-limiting process for successful gene expression
Endogenous or chemically-modified nuclear localization signal (NLS) are recognized by karyopherins, and the cargo is allowed to pass though the nuclear pore, with the aid of interactions with hydrophobic phenylalanine-glycine (FG)-rich domains inside the nuclear pore complex (NPC) [8,9,10]
The E(cyt) was impaired by the synchronization (Figure 1D). These results indicate that the hydroxyurea treatment blocked the NPC-independent nuclear delivery process; non-specific nuclear delivery concomitant with the temporal collapse of the nuclear membrane structure at mitosis
Summary
The nuclear delivery of plasmid DNA (pDNA) is a crucial rate-limiting process for successful gene expression. Endogenous or chemically-modified NLSs are recognized by karyopherins (i.e., importin α and/or β), and the cargo is allowed to pass though the nuclear pore, with the aid of interactions with hydrophobic phenylalanine-glycine (FG)-rich domains inside the NPC [8,9,10]. Another type of nuclear import carrier (i.e., Hikeshi) that is not a member of the conventional importin family has recently been identified as the nuclear import machinery of
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