Subcellular localization of EBNA1 and oriP that may support the high rate of exogenous gene transfer achieved using the EBV genetic elements

摘要

Recombinant DNA constructs containing the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene and oriP element shows various features including cytoplasm-to-nuclear transport as well as association with nuclear matrix and chromosomes, after being introduced into a rodent cell, while in a cell of human origin, the EBNA1 and oriP-containing vector also replicates in synchrony with chromosomal DNA duplication. We reported that the EBV-based plasmid vector with the artificial chromosome-like characteristics provides a useful means to analyze bioactivity in vitro and in vivo of various genes in mammals. Using multi-scale manipulation techniques, we have also analyzed intracellular localization and functions of the EBV-based plasmid vector. When fluorescence-labeled DNA containing the oriP sequence was transfected by lipofectamine (Invitrogen) into HeLaS3 cells or HeLaS3-derived transformant that had been engineered to express EBNA1, the extracellular-to-intracellular transfer of the DNA was not significantly affected by the presence or absence of EBNA1, as revealed by flowcytometric analysis. Because transfection with an oriP-containing expression vector resulted in remarkably higher gene expression in EBNA1 expressing cells in comparison with that with a plasmid vector lacking oriP, the findings suggest that the rates of transfection and gene expression are highly dependent on the efficiency of some intracellular events including intracytoplasmic-to-intranuclear transfer of plasmid rather than of extracellular-to-intracellular DNA transfer. Intranuclear behavior of EBNA1 and oriP was then analyzed by transfecting cells with EBNA1-AcGFP chimeric gene in combination with fluorescenated DNA with or without oriP followed by the on-substrate cell lysis analyses.