Efficient transfer of chromosome-based DNA constructs into mammalian cells

Oberle, Volker; Jong, Gary de; Drayer, Jan I.; Hoekstra, Dick

doi:10.1016/j.bbaexp.2003.12.003

Cited by 27 publications

(21 citation statements)

References 30 publications

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“…Ultrasound is also known to increase intracellular uptake of large molecules [41], including enhanced delivery of artificial chromosomes complexed with cationic lipids, which are as large as 1-2 m in size [42]. Increased intracellular delivery is believed to occur due to the formation of transient pores of nanometer to micron dimensions [25,43,44].…”

Section: Discussionmentioning

confidence: 99%

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

Deshpande

Prausnitz

2007

Journal of Controlled Release

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Ultrasound and poly(ethylenimine) (PEI) have each separately been shown to increase DNA transfection efficiency. This study tested the hypothesis that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. This in vitro study assessed transfection efficiency of two different DNA plasmids encoding green fluorescent protein and firefly luciferase in two different cells types, a primary culture of human aortic smooth muscle cells and an immortal line of human prostrate cancer cells. We found that ultrasound sonication increased transfection up to 18-fold, DNA complexation with PEI increased transfection up to 90-fold, and the combination of ultrasound and PEI synergistically increased transfection up to 200-fold, which resulted in reporter gene expression by 34% of cells. Kinetic measurements found that the effects of ultrasound alone acted quickly, whereas increased transfection by PEI either alone or in combination with ultrasound strongly benefited from a 4-h incubation with the DNA plasmid after sonication. Although serum reduced absolute expression levels, it did not affect the relative increase in transfection when ultrasound was added to PEI enhancement. Flow cytometry measurements showed that sonication increased intracellular uptake of labelled DNA complexed to PEI by 55% relative to PEI complexation alone. Electrophoresis assay showed no damage to DNA or PEI-DNA complexes after sonication. Overall, these results suggest that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection.

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Section: Discussionmentioning

confidence: 99%

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

Deshpande

Prausnitz

2007

Journal of Controlled Release

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“…As these artificial chromosomes are designed to carry large DNA inserts, these systems are not very suitable for use as vectors in gene therapy applications where, in most cases, only a few genes need to be expressed. Delivery of chromosomal vectors is relatively complicated, particularly because their large size (1Y2 mm), in comparison to plasmids (sizes generally within the nanometer range in condensed form), hampers cellular uptake via the endocytic route (120). Additionally, the structural stability of chromosomal vectors containing both DNA and proteins is inferior to that of pure plasmid DNA (120).…”

Section: Episomally Replicating Vectorsmentioning

confidence: 99%

Plasmid Engineering for Controlled and Sustained Gene Expression for Nonviral Gene Therapy

et al. 2006

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Gene therapy requires the introduction of genetic material in diseased cells with the aim of treating or ultimately curing a disease. Since the start of gene therapy clinical trials in 1990, gene therapy has proven to be possible, but studies to date have highlighted the difficulty of achieving efficient, specific, and long-term transgene expression. Efforts to improve gene therapy strategies over the past years were mainly aimed at solving the problem of delivery, without paying much attention to the optimization of the expression cassette. With the current understanding of the eukaryotic transcription machinery and advanced molecular biology techniques at our disposition, it has now become possible to create custom-made transgene expression cassettes optimized for gene therapy applications. In this review, we will discuss several strategies that have been explored to improve the level and duration of transgene expression, to increase control over expression, or to restrict transgene expression to specific cell types or tissues. Although still in its infancy, such strategies will eventually lead to improvement of nonviral gene therapy and expansion of the range of possible therapeutic applications.

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“…This event disconnects the puromycin resistance gene from its promoter and replaces it with the promoterless neomycin resistance gene, which in this way acquires the SV40 promoter. [18][19][20][21][22]. These experiments led us to choose to use the Superfect reagent (Qiagen) and we optimized the delivery of isolated metaphase chromosomes, originating from cell lines carrying tACs, into a mES cell line, R1, which was kindly provided by Prof. Andras Nagy [23].…”

Section: Resultsmentioning

confidence: 99%

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe’s disease in the Twitcher mouse

Katona

Sinkó

Holló

et al. 2008

Cell. Mol. Life Sci.

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Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.

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Efficient transfer of chromosome-based DNA constructs into mammalian cells

Cited by 27 publications

References 30 publications

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

Plasmid Engineering for Controlled and Sustained Gene Expression for Nonviral Gene Therapy

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe’s disease in the Twitcher mouse

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