An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus

Sengupta, Ranjita; Mukherjee, Chandreyee; Sarkar, Nandita; ZhiHong, Sun; Lesnik, Jacob; Huang, Joseph; Lü, Biao

doi:10.1186/s12575-016-0044-z

Cited by 7 publications

(6 citation statements)

References 17 publications

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“…Pseudotyping is often used in the production of recombinant viruses and involves packaging the genetic components of the virus (DNA or RNA) with envelope proteins derived from a different virus. [28][29][30] This method allows one to select viral envelope proteins to alter host tropism, which may result in enhanced infection of the recombinant virus. 31,32 The G glycoprotein of the vesicular stomatitis virus glycoprotein (VSVG) is frequently used for viral pseudotyping, owing to its broad tropism and high efficacy in transduction, and in fact most of the retroviruses approved for clinical trials are pseudotyped with VSVG.…”

Section: Introductionmentioning

confidence: 99%

Pseudotyping exosomes for enhanced protein delivery in mammalian cells

Meyer

Losacco

Stickney

et al. 2017

IJN

Self Cite

119

100

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Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells.

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Section: Introductionmentioning

confidence: 99%

Pseudotyping exosomes for enhanced protein delivery in mammalian cells

Meyer

Losacco

Stickney

et al. 2017

IJN

Self Cite

119

100

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“…Previously we and others demonstrated that the vesicular stomach virus envelope glycoprotein (VSVG)-pseudotyped lentiviruses have a super high infection rate of ~100% [ 36 , 37 ]. In this study we chose VSVG-pseudotyping protocol for lenti-Cre virus production, and examined whether VSVG-pseudotyped lenti-Cre-GFP could deliver, express and subsequently edit the knockin floxed-RFP-Puro in reporter cells.…”

Section: Resultsmentioning

confidence: 99%

“…Previously we had developed a system for delivering various genes into cells using either lentiviral or AAV particles [ 36 , 38 ]. We initially selected the lentiviral system because of its favorable features: (1) Lentiviral transduction is an efficient way to create a stable cell line expressing Cre in almost any mammalian cell type including stem cells and non-dividing cells.…”

Section: Resultsmentioning

confidence: 99%

“…(2) Lentiviral system can be customized with a Cumate-inducible promoter driving expression of Cre, enabling scientists to follow biological changes induced by Cre in real time, thus expanding its application (Additional file 2 : Figure S1). (3) There is a newly available integrase-defective lentiviral packaging system, which enables transduction of larger lentiviral constructs without undesired integration of the lenti construct into the genome [ 36 ]. In contrast, AAV vectors have a smaller gene packaging size of ~ 3.7 kb, which make them unsuitable for building all-in-one inducible Cre (~7.0 kb in size).…”

Section: Resultsmentioning

confidence: 99%

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Viral Cre-LoxP tools aid genome engineering in mammalian cells

et al. 2017

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Background: Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. Results: We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions.

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“…Images of living cells were typically taken using fluorescence microscopy as reported [33]. To show the intensity of GFP expression, both fluorescent and phase contrast images were recorded.…”

Section: Live Fluorescence Microscopymentioning

confidence: 99%

A Novel Genetic Circuit Supports Laboratory Automation and High Throughput Monitoring of Inflammation in Living Human Cells

Duong¹,

Curley²,

Mai³

et al. 2019

Cell Signalling - Thermodynamics and Molecular Control

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Genetically encoded reporter circuits have been revolutionizing our ability to monitor, manipulate, and visualize specific cellular responses to a variety of environmental stimuli. However, the development of genetic circuits that enable both high throughput (HTP) application and laboratory automation remains challenging. In this report, we describe a novel dual-reporter circuit that utilizes a secretory Gaussia luciferase (Gluc) and a green fluorescent protein (GFP) for monitoring inflammatory signaling, a fundamental process in many life events. We designed and built this genetic circuit into a simple adeno-associated viral (AAV) vector, which is suitable for both simple transfection and efficient transduction protocols. We demonstrated high sensitivity and specificity of this new circuit and its ability to monitor a broad range of inflammatory response in various human cell models. Importantly, this novel system is simple, robust, and readily adaptable to HTP applications and laboratory automation including fluorescence activated cell sorting (FACS) and microplate reader analysis. By combining both GFP and Gluc in one genetic circuit, our new dualreporter circuit provides an easy and powerful tool for monitoring and quantifying inflammatory signals in various mammalian cells.

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An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus

Cited by 7 publications

References 17 publications

Pseudotyping exosomes for enhanced protein delivery in mammalian cells

Pseudotyping exosomes for enhanced protein delivery in mammalian cells

Viral Cre-LoxP tools aid genome engineering in mammalian cells

A Novel Genetic Circuit Supports Laboratory Automation and High Throughput Monitoring of Inflammation in Living Human Cells

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