Polyswitch Lentivectors: “All-in-One” Lentiviral Vectors for Drug-Inducible Gene Expression, Live Selection, and Recombination Cloning

Giry-Laterrière, Marc; Cherpin, Ophélie; Kim, Yong Sik; Jensen, Jan; Salmon, Patrick

doi:10.1089/hum.2010.179

Cited by 25 publications

(19 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Stable populations of NMuMG cells expressing the blasticidine-resistant marker together with TFAP2A-FLAG under a doxycycline-inducible promoter were obtained with the pCLX expression system [26]. Lentiviral particles were produced in HEK293-LV cells using the helper vectors pMDL, pREV and the envelope-encoding vector pVSV.…”

Section: Methodsmentioning

confidence: 99%

TFAP2A is a component of the ZEB1/2 network that regulates TGFB1-induced epithelial to mesenchymal transition

et al. 2017

View full text Add to dashboard Cite

BackgroundThe transition between epithelial and mesenchymal phenotypes (EMT) occurs in a variety of contexts. It is critical for mammalian development and it is also involved in tumor initiation and progression. Master transcription factor (TF) regulators of this process are conserved between mouse and human.MethodsFrom a computational analysis of a variety of high-throughput sequencing data sets we initially inferred that TFAP2A is connected to the core EMT network in both species. We then analysed publicly available human breast cancer data for TFAP2A expression and also studied the expression (by mRNA sequencing), activity (by monitoring the expression of its predicted targets), and binding (by electrophoretic mobility shift assay and chromatin immunoprecipitation) of this factor in a mouse mammary gland EMT model system (NMuMG) cell line.ResultsWe found that upon induction of EMT, the activity of TFAP2A, reflected in the expression level of its predicted targets, is up-regulated in a variety of systems, both murine and human, while TFAP2A’s expression is increased in more “stem-like” cancers. We provide strong evidence for the direct interaction between the TFAP2A TF and the ZEB2 promoter and we demonstrate that this interaction affects ZEB2 expression. Overexpression of TFAP2A from an exogenous construct perturbs EMT, however, in a manner similar to the downregulation of endogenous TFAP2A that takes place during EMT.ConclusionsOur study reveals that TFAP2A is a conserved component of the core network that regulates EMT, acting as a repressor of many genes, including ZEB2.ReviewersThis article has been reviewed by Dr. Martijn Huynen and Dr. Nicola Aceto.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-017-0180-7) contains supplementary material, which is available to authorized users.

show abstract

Section: Methodsmentioning

confidence: 99%

TFAP2A is a component of the ZEB1/2 network that regulates TGFB1-induced epithelial to mesenchymal transition

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Lentiviral vector stocks were generated using transient transfection of HEK 293T/17 cells with the specific lentivector transfer plasmid, the psPAX2 plasmid encoding gag/pol and the pCAG-VSVG envelope plasmid, as previously described. 42,43 Lentivector titer was performed using transduction of HT-1080 cells followed by flow cytometry quantification of GFP-positive (or mCherry positive) cells 5 days after infection, as previously described. 42,43 …”

Section: Methodsmentioning

confidence: 99%

“…42,43 Lentivector titer was performed using transduction of HT-1080 cells followed by flow cytometry quantification of GFP-positive (or mCherry positive) cells 5 days after infection, as previously described. 42,43 …”

Section: Methodsmentioning

confidence: 99%

Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

Myburgh

Cherpin

Schlaepfer

et al. 2014

Molecular Therapy - Nucleic Acids

Self Cite

View full text Add to dashboard Cite

Gene knockdown using micro RNA (miRNA)-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp), positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC); incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.

show abstract

“…The different expression constructs were generated using the Gateway recombination cloning technology on lentiviral destination vectors (Giry-Laterriè re et al, 2011a, 2011b. In a first type of vector, a single expression unit is composed of one promoter driving one gene.…”

Section: Plasmidsmentioning

confidence: 99%

Wnt Signaling Regulates Multipolar-to-Bipolar Transition of Migrating Neurons in the Cerebral Cortex

et al. 2015

Self Cite

View full text Add to dashboard Cite

The precise timing of pyramidal cell migration from the ventricular germinal zone to the cortical plate is essential for establishing cortical layers, and migration errors can lead to neurodevelopmental disorders underlying psychiatric and neurological diseases. Here, we report that Wnt canonical as well as non-canonical signaling is active in pyramidal precursors during radial migration. We demonstrate using constitutive and conditional genetic strategies that transient downregulation of canonical Wnt/β-catenin signaling during the multipolar stage plays a critical role in polarizing and orienting cells for radial migration. In addition, we show that reduced canonical Wnt signaling is triggered cell autonomously by time-dependent expression of Wnt5A and activation of non-canonical signaling. We identify ephrin-B1 as a canonical Wnt-signaling-regulated target in control of the multipolar-to-bipolar switch. These findings highlight the critical role of Wnt signaling activity in neuronal positioning during cortical development.

show abstract

Polyswitch Lentivectors: “All-in-One” Lentiviral Vectors for Drug-Inducible Gene Expression, Live Selection, and Recombination Cloning

Cited by 25 publications

References 49 publications

TFAP2A is a component of the ZEB1/2 network that regulates TGFB1-induced epithelial to mesenchymal transition

TFAP2A is a component of the ZEB1/2 network that regulates TGFB1-induced epithelial to mesenchymal transition

Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

Wnt Signaling Regulates Multipolar-to-Bipolar Transition of Migrating Neurons in the Cerebral Cortex

Contact Info

Product

Resources

About