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Open AccessCommunication
Pharmaceutics 2015, 7(2), 64-73; doi:10.3390/pharmaceutics7020064

Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells

1
Faculty of Pharmaceutical Science, Hokkaido University, Kita-12, Nishi-6, Kita-ku, 060-0812 Sapporo, Japan
2
PlasmidFactory GmbH & Co. KG. Meisenstraße 96, D-33607 Bielefeld, Germany
These authors contributed equally to this work.
Hidetaka Akita and Dai Kurihara equally contribute to this study. The authors are listed alphabetically in accordance with family name.
*
Authors to whom correspondence should be addressed.
Academic Editor: Keiji Itaka
Received: 10 May 2015 / Revised: 30 May 2015 / Accepted: 4 June 2015 / Published: 9 June 2015
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
View Full-Text   |   Download PDF [802 KB, uploaded 9 June 2015]   |  

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. View Full-Text
Keywords: DNA; nuclear transfer; size; microinjection DNA; nuclear transfer; size; microinjection
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Akita, H.; Kurihara, D.; Schmeer, M.; Schleef, M.; Harashima, H. Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells. Pharmaceutics 2015, 7, 64-73.

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