Lentiviral Vector Design for Multiple shRNA Expression and Durable HIV-1 Inhibition

Brake, Olivier ter; Hooft, Karen 't; Liu, Ying Poi; Centlivre, Mireille; Eije, Karin J. von; Berkhout, Ben

doi:10.1038/sj.mt.6300382

Cited by 218 publications

(247 citation statements)

References 48 publications

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“…Several viruses have been engineered to efficiently deliver genetic cargos into mammalian cells (4). Retro-and lentiviruses are not ideal due to their highly recombinogenic single-stranded RNA genome (5). In fact, a recent attempt to develop a lentiviral vector for UAA mutagenesis in mammalian cells was limited to a single tRNA expression cassette, and required multiple vectors to deliver all of the required genetic elements, significantly compromising its efficiency and utility (6).…”

Section: Resultsmentioning

confidence: 99%

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Chatterjee

Xiao

Bollong

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

109

132

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Here we report the development of a baculovirus-based delivery system that enables the efficient incorporation of unnatural amino acids into proteins in mammalian cells. We have exploited the large cargo-capacity (>30 kb) and stability of the double-stranded DNA genome of baculovirus to deliver to a variety of cell types all of the components required to genetically incorporate novel amino acids. These include the engineered tRNA/aminoacyl-tRNA synthetase pair and the nonsense mutant of the target gene. Mammalian cell transduction efficiency of baculovirus was significantly improved by incorporating genetic elements from mammalian viruses. Two polyspecific tRNA/aminoacyl-tRNA synthetase pairs were inserted into this expression system, enabling the site-specific incorporation of a variety of unnatural amino acids with novel chemical and biological properties into proteins.genetic code | viral vector | nonstandard amino acid | orthogonal

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Section: Resultsmentioning

confidence: 99%

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Chatterjee

Xiao

Bollong

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

109

132

View full text Add to dashboard Cite

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“…However, care must be taken to space each shRNA-expression cassette because the simple combination of the expression cassettes and the repeat sequence may result in reduced activity of each shRNA or even deletion of some shRNA. 47 …”

Section: Discussionmentioning

confidence: 99%

“…22,23 The multiple shRNA strategy has been investigated in the context of human immunodeficiency virus-1 treatment and shown to successfully overcome viral escape and mutation. 24,25 For example, Brake et al 26 showed that human immunodeficiency virus-1 could escape from a single shRNA attack but not from four shRNAs that were simultaneously expressed in a cell. A multiple shRNA strategy was also used to silence hepatitis C or B virus and superior inhibition of viral replication was observed.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

Payne

Sun

et al. 2010

Cancer Gene Ther

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RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.

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“…As REs usually require addition of a minimal CMV promoter to achieve high expression levels, we substituted the CMV promoter-driving DsRed with the hPGK promoter to avoid the potential of homologous recombination due to repeated sequences, as previously reported for lentiviral vectors. 29 We also replaced EGFP with ZsG-DR, a brighter and destabilized (t 1/2 ¼ 8 --12 h) green fluorescent protein to better capture the kinetics of gene expression. Finally, we cloned the promoter for the well-known early myogenic marker, aSMA upstream of ZsG-DR, to monitor smooth muscle cell maturation in response to TGF-b1 treatment.…”

Section: Generation Of Shlvdp Vectormentioning

confidence: 99%

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

et al. 2012

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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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Lentiviral Vector Design for Multiple shRNA Expression and Durable HIV-1 Inhibition

Cited by 218 publications

References 48 publications

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

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

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