9]Engineering 100- to 300-kb DNA as persisting extrachromosomal elements in human cells using human artificial episomal chromosome system

Abstract

Publisher Summary This chapter focuses on the mechanism of engineering 100- to 300-kb DNA as persisting extrachromosomal elements in human cells by using human artificial episomal chromosome system. It also discusses the development of a human artificial episomal chromosome (HAEC) system for cloning and functional analysis of large DNA fragments in human cells. The HAEC system is based on the latent replication origin ( oriP ) of the Epstein–Barr virus (EBV), a large human herpes virus. This system permits the propagation and stable maintenance of large DNA inserts, ranging from 60–330 kb, in human cells as artificial episomal chromosomes. A pilot HAEC library containing 1500 independent colonies with insert sizes ranging from 100–200 kb has been constructed, which covers approximately 10% of the human genome. The HAEC DNA established at 50-100 copies per cell could be recovered as large intact supercoiled DNA. It was found during the experiment that most cell transformants carried one or two distinct HAEC clones and individual HAECs carried single human genomic inserts. The large genomic DNA inserts were found to be genetically distinct and derived from different regions of the human genome and appeared genetically stable. The multicopy DNA, clonality, and stability of the HAEC system make it suitable for cloning various large human sequences in human cells. It provides an experimental approach for the functional study of large DNA regions in human cells and potentially for episome-based gene therapy.