Nuclear Entry of DNA and Transgene Expression in Dividing and Non-dividing Cells.

Abstract

Plasmid DNA (pDNA) must be delivered into the nucleus for transgene expression in mammalian cells. The entry may happen passively during the nuclear envelope breakdown and reformation in dividing cells or actively through the nuclear pore complexes. The goal of this study was to investigate the relative importance of these two pathways for pDNA nuclear entry and subsequent gene expression. To measure nuclear entry of pDNA encoding enhanced green florescence protein (EGFP) in electrotransfected cells, we developed a sensitive technique for quantitative analysis of pDNA in the nuclei, based on a hybridization probe for pDNA detection at the single molecule level and automatic image analysis. In matched experiments, we used an mRNA targeted hybridization probe to quantify reporter mRNA expression per cell, and flow cytometry to quantify expression of EGFP. We discovered two distinct patterns of pDNA distribution in the nuclei: punctate and diffuse, which were dominant in arrested and unarrested cells, respectively. The cell cycle arrest decreased diffuse pDNA and increased punctate pDNA. Its net effect was a decrease in the total intranuclear pDNA. Additionally, the cell cycle arrest increased the reporter mRNA synthesis but had no substantial impact on reporter protein expression. Results from the study demonstrated that the efficient nuclear entry of pDNA during cell division did not necessarily lead to a high level of transgene expression. They also suggested that the punctate pDNA was more transcriptionally active than diffuse pDNA in the nuclei. These data will be useful in future studies for understanding mechanisms of nonviral gene delivery.