Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

Abstract

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 B10-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 -methylguanine-DNA 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.

miR-99a/100∼125b tricistrons regulate hematopoietic stem and progenitor cell homeostasis by shifting the balance between TGFβ and Wnt signaling

“…Briefly, we inserted eight to 16 specific target sites of each miRNA as an artificial 39 UTR behind the sense SFFV-driven mCherry cassette, while an antisense CMV-driven GFP-IRES-Puro served as selection marker (pLBid-GIP/C vector) (Supplemental Fig. S5C,D; Maetzig et al 2010). The SP-sc control sequence was adapted from Loya et al (2009).…”

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

“…Vector-derived copy numbers were determined by real-time PCR as described previously (18,19). A total of 100 ng/2 ml genomic DNA prepared from the above step were added to the 13-ml RQ-PCR mix containing 7.5 ml SYBRTaq mix with 1 ml primer mix for woodchuck hepatitis virus posttranscriptional regulatory element, 59-GAGGAGTTGTGGCCCGTTGT-39 (forward), and 59-TGACA-GGTGGTGGCAATGCC-39 (reverse); or polypyrimidine tract binding protein 2, 59-TCTCCATTCCCTATGTTCATGC-39 (forward), and 59-GTTCCCGCAGAATGGTGAGGTG-39 (reverse), adjusting the volume to 13 ml with PCR-grade nuclease-free water.…”

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

“…To isolate genomic DNA from cells, the QIAamp DNA blood kit (Qiagen) was used according to the manufacturer's instructions. Vector copy numbers of transduced cells were determined by quantitative PCR on an Applied Biosystems (Darmstadt, Germany) Step One Plus real-time PCR system using the QuantiTect SYBR green kit (Qiagen) with a reference sample known to contain 9 integrations (clone B, internal standard; kindly provided by U. Modlich, Hannover, Germany) and primers for the vector-specific PRE and the PTBP2 intron (evolutionarily conserved PTBP2 intronic sequence [28]) (19). From PCR efficiencies and by cross-point deviations from the reference sample, the vector copy numbers of unknown samples were determined according to the work of Pfaffl (27).…”