Generation of a conditionally neo(r)-containing retroviral producer cell line: effects of neo(r) on retroviral titer and transgene expression

Details

Serval ID
serval:BIB_0AE22EF29B81
Type
Article: article from journal or magazin.
Collection
Publications
Title
Generation of a conditionally neo(r)-containing retroviral producer cell line: effects of neo(r) on retroviral titer and transgene expression
Journal
Gene Ther
Author(s)
Wildner O., Candotti F., Krecko E. G., Xanthopoulos K. G., Ramsey W. J., Blaese R. M.
ISSN
0969-7128 (Print)
ISSN-L
0969-7128
Publication state
Published
Issued date
05/1998
Volume
5
Number
5
Pages
684-91
Language
english
Notes
Wildner, O
Candotti, F
Krecko, E G
Xanthopoulos, K G
Ramsey, W J
Blaese, R M
eng
Research Support, Non-U.S. Gov't
England
Gene Ther. 1998 May;5(5):684-91.
Abstract
We have developed a method for generating high-titer retroviral producer cell lines conditionally containing a neomycin resistance gene (neo(r)) based on the Cre/loxP system. For this, a bicistronic retroviral splicing vector carrying the green fluorescence protein (GFP) and a marker gene cassette consisting of internal ribosome entry site (IRES) and neo(r) flanked by loxP sites, was constructed and conveniently used to generate a G418 resistant vector producer cell line. Following titer determination and verification of the biological activity of the retroviral supernatants, the selectable expression cassette which was no longer required was excised from the provirus by transient Cre expression using an adenoviral vector. This strategy led to precise excision of neo(r) and generation of retroviral supernatants containing functional 'neo-less' retroviral particles without detrimental effects on the high vector titers found in the parental neo(r)-containing producer lines. GFP expression was significantly increased after the excision of neo(r), in both the producer lines and retrovirally transduced target cells. Reintroduction of neo(r) did not alter GFP expression, suggesting that the neo(r) gene and/or its gene product per se are not acting as a transcriptional silencer.
Keywords
Adenoviridae/*genetics, Animals, Anti-Bacterial Agents, Blotting, Northern, Blotting, Southern, Cell Line, Drug Resistance/genetics, Flow Cytometry, Gene Expression Regulation, Viral, Genetic Markers, *Genetic Vectors, Green Fluorescent Proteins, Humans, Indicators and Reagents/metabolism, Integrases/genetics/metabolism, Luminescent Proteins/metabolism, Neomycin, RNA, Messenger/isolation & purification, RNA, Viral/analysis, Retroviridae/*genetics, Transgenes, *Viral Proteins
Pubmed
Open Access
Yes
Create date
01/11/2017 10:29
Last modification date
20/08/2019 12:32
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