Improvements in the Epstein-Barr-based shuttle vector system for direct cloning in human tissue culture cells

Richard A. Swirski, David Van Den Berg, Andrew J M Murphy, Claire M. Lambert, Errol C. Friedberg, Robert T. Schimke

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

We describe an Epstein-Barr virus (EBV)-based expression vector system for human cells that results in high transfection efficiencies (1-30%) in permissive human cell lines. The vector can replicate episomally for up to 6 months in a nonrearranged form. Human fibroblast cell lines are first rendered permissive by stable integration of an Epstein-Barr nuclear antigen-1 expression vector (CMV-EBNA). The EBV vector is a modification of that of B. Sugden et al. (1985, Mol. Cell. Biol. 5, 410) and its functional elements, when introduced into "permissive" cells, include the EBV origin of plasmid replication, a drug resistance gene for selection in human cells (hygromycin), and bacterial sequences for maintenance in Escherichia coli. We have introduced an expression cassette for the production of high levels of mRNA from the RSV LTR such that transcription is in the same direction as replication fork progression. The high transfection frequency and maintenance in an episomal form allow for efficient transfection of complete cDNA libraries and for ready recovery of DNA sequences encoding selectable phenotypes.

Original languageEnglish (US)
Pages (from-to)133-142
Number of pages10
JournalMethods
Volume4
Issue number2
DOIs
StatePublished - Aug 1992

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'Improvements in the Epstein-Barr-based shuttle vector system for direct cloning in human tissue culture cells'. Together they form a unique fingerprint.

  • Cite this