Generation of a conditionally neo(r)-containing retroviral producer cell line: effects of neo(r) on retroviral titer and transgene expression.
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.
Wildner
O
O
Clinical Gene Therapy Branch/National Human Genome Research Institute, National Institute of Health, Bethesda, MD 20892-1851, USA.
Candotti
F
F
Krecko
E G
EG
Xanthopoulos
K G
KG
Ramsey
W J
WJ
Blaese
R M
RM
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000900:Anti-Bacterial Agents; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D009355:Neomycin; D012333:RNA, Messenger / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D019076:Transgenes; D014764:Viral Proteins
D000900:Anti-Bacterial Agents; D005819:Genetic Markers; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases; D009355:Neomycin