Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector

Tian, Jun; Andreadis, Stelios T.

doi:10.1038/gt.2009.46

Cited by 56 publications

(74 citation statements)

References 41 publications

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“…Generally, internal transcription units are inserted in the lentiviral vector without poly(A) signal to avoid truncation of the full-length viral genome (Shimotohno and Temin 1981), although inclusion of an internal poly(A) signal can enhance transgene expression in virus-infected target cells (Shimotohno and Temin 1981;Maxwell et al 1991;Narita et al 2000;Hager et al 2008;Tian and Andreadis 2009). In accordance with the findings of Hager et al (2008), we found that an internal poly(A) signal reduced the transduction titer with z1 log.…”

Section: Discussionsupporting

confidence: 85%

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

Liu¹,

Vink²,

Westerink³

et al. 2010

RNA

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RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.

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Section: Discussionsupporting

confidence: 85%

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

Liu¹,

Vink²,

Westerink³

et al. 2010

RNA

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show abstract

“…22 A constitutive promoter drives expression of one marker gene (for example, DsRed) and is used to measure transduction efficiency. The promoter of interest drives expression of another marker (for example, green fluorescent protein (EGFP)) and is used to monitor the biological process of interest.…”

Section: Generation Of Shlvdp Vectormentioning

confidence: 99%

“…These sequences were cloned in trans to the viral LTRs to avoid premature termination of viral mRNA by the inserted polyAs during virus production in packaging cells as reported previously. 22 The resulting shRNA encoding LentiViral Dual Promoter vectors were termed shLVDP vectors.…”

Section: Lentiviral Vector Construction Strategy and Lentiviral Vectomentioning

confidence: 99%

“…After 3 days, the cells were imaged and 2 days later they were procesed for flow cytometry as described previously. 22 Cell transfection and gene knockdown 293T cells (8 Â 10 5 per well) were seeded in 6-well tissue culture-treated plates and transfection was done using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. Transfection was performed for 24 h and cells were replenished with fresh medium.…”

Section: Infection and Differentiation Protocolmentioning

confidence: 99%

“…The second encodes for discosoma red fluorescent protein (DsRed)-Express under a constitutive promoter for example, human phosphoglycerate kinase promoter (hPGK) and is used to assess gene transfer efficiency as well as for data normalization to obtain quantitative data independent of the gene transfer efficiency. 21,22 Finally, the third unit encodes for shRNA under a tetracycline-regulatable H1 promoter in the viral LTR. This vector allows shRNA expression and at the same time provides quantitative real-time monitoring of the effect of gene knockdown on cellular phenotype.…”

Section: Introductionmentioning

confidence: 99%

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A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

et al. 2012

Self Cite

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Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiation differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time, dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promoter vector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown. In this vector, the activity of the alpha-smooth muscle actin (aSMA) promoter was measured by the expression of a destabilized green fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma red fluorescent protein was used to measure transduction efficiency and to normalize aSMA promoter activity; and shRNA was encoded by a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titer allowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-b1 or Rho signaling pathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such as Smad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increased aSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenic differentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNA expression enabled temporal control of gene knockdown and provided dynamic information on the effect of different genes on myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to decipher gene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.

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New function of a well‐known promoter: Enhancer activity of minimal CMV promoter enables efficient dual‐cassette transgene expression

Boateng-Antwi

Lin

Ren

et al. 2021

The Journal of Gene Medicine

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Background: Co-expression of multiple genes in single vectors has achieved varying degrees of success by employing two promoters and/or application of viral 2Apeptide or the internal ribosome entry-site (IRES). However, promoter interference, potential functional-interruption of expressed-proteins by 2A-generated residual peptides or weaker translation of IRES-mediated downstream genes has curtailed their utilization. Thus, there is the need for single vectors that robustly express multiple proteins for enhanced gene therapy applications.Methods: We engineered lentiviral-vectors for dual-cassette expression of green fluorescent protein and mCherry in uni-or bidirectional architectures using the short-version (Es) of elongation factor 1α (EF) promoter and simian virus 40 promoter (Sv). The regulatory function of a core fragment (cC) from human cytomegalovirus promoter was investigated with cell-lineage specificity in NIH3T3 (fibroblast) and hematopoietic cell lines U937 (monocyte/macrophage), LCL (lymphoid), DAMI (megakaryocyte) and MEL (erythroid). Results:The cC element in reverse-orientation not only boosted upstream Es promoter to levels comparable to full-length EF in DAMI, U937 and 3T3 cells, but also blocked the suppression of downstream Sv promoter by Es in U937 and 3T3 cells with further improved Sv activity in DAMI cells. Such lineage-restricted up-regulation is likely attributed to two protein-binding domains of cC and diverse expression of related factors in different cell types for enhancer and terminator activities, but not spacing function.Conclusions: Such a newly developed dual-cassette vector could be advantageous, particularly in hematopoietic cell-mediated gene/cancer therapy, by allowing for independent and robust co-expression of therapeutic gene(s) and/or a selectable gene or imaging marker in the same cells.

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Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector

Cited by 56 publications

References 41 publications

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

New function of a well‐known promoter: Enhancer activity of minimal CMV promoter enables efficient dual‐cassette transgene expression

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