期刊
GENE THERAPY
卷 17, 期 3, 页码 400-411出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/gt.2009.129
关键词
bidirectional; lentivirus; gammaretrovirus; Nodamuravirus; O-6-methylguanine-DNA methyltransferase; Cre
类别
资金
- German Ministry for Research and Education
- DAAD (German-Chinese junior research groups)
- Deutsche Forschungsgemeinschaft (DFG) [SFB738]
- European Union [LSHB-CT-2004-005242, LSHB-CT-2006-018933]
- German National Merit foundation
- Else Kroner Fresenius Stiftung
Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were similar to 10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O-6-methylguanineDNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression. Gene Therapy (2010) 17, 400-411; doi: 10.1038/gt.2009.129; published online 22 October 2009
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