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

doi:10.1016/1046-2023(92)90045-A

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 journal › Article › peer-review

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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 language	English (US)
Pages (from-to)	133-142
Number of pages	10
Journal	Methods
Volume	4
Issue number	2
DOIs	https://doi.org/10.1016/1046-2023(92)90045-A
State	Published - Aug 1992

ASJC Scopus subject areas

Molecular Biology
Biochemistry, Genetics and Molecular Biology(all)

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

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.",

author = "Swirski, {Richard A.} and {Van Den Berg}, David and Murphy, {Andrew J M} and Lambert, {Claire M.} and Friedberg, {Errol C.} and Schimke, {Robert T.}",

note = "Funding Information: We thank Dr. W. Sugden for his vectors ~220.2 and pHyg and the anti-EBNA-1 antibody. This work was supported by a grant from the National Cancer Institute (CAHG 54346) and a postdoctoral fellowship (PF-3314) awarded A.J.M.M. from the American Cancer Society.",

year = "1992",

month = aug,

doi = "10.1016/1046-2023(92)90045-A",

language = "English (US)",

volume = "4",

pages = "133--142",

journal = "Methods in Enzymology",

issn = "0076-6879",

publisher = "Academic Press Inc.",

number = "2",

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T1 - Improvements in the Epstein-Barr-based shuttle vector system for direct cloning in human tissue culture cells

AU - Swirski, Richard A.

AU - Van Den Berg, David

AU - Murphy, Andrew J M

AU - Lambert, Claire M.

AU - Friedberg, Errol C.

AU - Schimke, Robert T.

N1 - Funding Information: We thank Dr. W. Sugden for his vectors ~220.2 and pHyg and the anti-EBNA-1 antibody. This work was supported by a grant from the National Cancer Institute (CAHG 54346) and a postdoctoral fellowship (PF-3314) awarded A.J.M.M. from the American Cancer Society.

PY - 1992/8

Y1 - 1992/8

N2 - 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.

AB - 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.

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

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