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

Tokuko Haraguchi,Tomoko Shindo,Takako Koujin,Yuji Chikashige,Tatsuo Fukagawa,Şükriye Bilir,Kohei Nishimura,Shouhei Kobayashi,Shinsuke Shibata,Haruhiko Asakawa,Yasushi Hiraoka,Hiroko Osakada,Chie Mori,Yasushi Okada,Yasuhiro Hirano

doi:10.1038/s42003-022-03021-8

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.

Highlights

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA
When the DNA plasmid enters the cytosol after endosome rupture, it is visualized by binding to GFP-LacI, forming fluorescent puncta that are detected using fluorescence microscopy (Fig. 1a; arrows, Fig. 1b)
The GFP-LacI-positive puncta colocalized with the LEM domain nuclear envelope (NE) protein emerin and Lem[2] (Supplementary Fig. 1a, b). These puncta were colocalized with lamin B receptor (LBR) and calnexin (CNX) but not with lamin B1 and early endosome antigen 1 (EEA1) (Supplementary Fig. 1a, b). These results suggested that they were surrounded by an NE-like membrane that originated from the endoplasmic reticulum (ER) membrane in the cytosol after being released from the endosome, which is consistent with a report demonstrating that DNA-bead complexes become wrapped with NE-like membranes within 5–10 min after endosome rupture[8]

Summary

Introduction

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. 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. BAF is a DNA-binding protein[21,22,23,24] involved in NE reformation at the telophase of mitosis[25,26,27] It binds to foreign DNA, such as transfected DNA and viral DNA, in the cytosol[8,21,28,29], where it assembles the NE-like membrane containing LEM domain proteins, such as emerin, around the transfected DNA8

Methods

Results

Conclusion