Transfection by Cationic Liposomes Using Simultaneous Single Cell Measurements of Plasmid Delivery and Transgene Expression

Tseng, Wen‐Chi; Haselton, Frederick R.; Giorgio, Todd D.

doi:10.1074/jbc.272.41.25641

Cited by 170 publications

(103 citation statements)

References 26 publications

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“…It had been shown previously that the uptake of lipoplexes prepared with fluorescently labeled plasmid DNA could be measured by flow cytometry. 7,22,23 We initially determined the extent of fluorescence detected by this assay that is due to cell surface-associated SPLP compared with internalized SPLP. Since internalization of cationic lipid-formulated DNA is via the energydependent process of endocytosis, 7,8,10 we hypothesized that incubating cells with formulated fluorescent plasmid at 4°C would permit interaction at the cell surface but prevent endocytosis.…”

Section: Resultsmentioning

confidence: 99%

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

et al. 1999

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Cationic lipid-based delivery systems such as lipoplexes or aphidicolin exhibited 20-fold lower reporter gene activity stabilized plasmid-lipid particles (SPLP) represent a safer than asynchronous control cells upon incubation with lipoalternative to viral systems for gene therapy applications.plexes. When cells arrested in the G1 phase were allowed We studied the impact of cell cycle status on the efficiency to proceed though the cell cycle in the presence of the lipoof transfection of human ovarian carcinoma tumor cells plex or SPLP, transgene expression was found to coincide using two cationic-lipid based delivery systems. Cells with the transition of cells from the G2/M phase into the arrested in the G1 phase of the cell cycle by treatment with G1 phase of the subsequent cell cycle. In addition, higher aphidicolin were compared with an asynchronous dividing levels of reporter gene expression were observed when the population of cells. Treatment of the cells with aphidicolin cells were incubated with lipoplexes or SPLP during, or just had no effect on the rate of internalization of the lipid forbefore, mitosis. These results suggest that it may be possmulated DNA or on the level of gene expression observible to augment cationic lipid-mediated transfection by able in stably transfected cells. However, cells treated with manipulating the cell cycle status of the target cells.

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Section: Resultsmentioning

confidence: 99%

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

et al. 1999

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“…Traditionally, nonviral vectors are formed by complexation of plasmid DNA with cationic lipids (e.g., Lipofectamine) or cationic polymers (e.g., polyethylenimine, PEI), which reduces the negative surface charge, protects against degradation, and facilitates cellular internalization and intracellular trafficking of the plasmid. In vitro studies typically employ bolus addition of these complexes to the media, resulting in internalization of approximately 20-50% of this plasmid (Tseng et al, 1997;Varga et al, 2001). Alternative delivery strategies have shown that increasing the concentration of DNA in the cellular microenvironment can increase transfection (Luo and Saltzman, 2000).…”

Section: Introductionmentioning

confidence: 99%

Gene delivery through cell culture substrate adsorbed DNA complexes

Bengali

Pannier

Segura

et al. 2005

Biotech & Bioengineering

130

187

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Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serumadsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-coglycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering.

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“…Lipofections are sensitive to a number of variables; reagent concentrations are among the most critical. 21 Dose responses for plasmid expression tend to have an optimum balanced between low uptake at low reagent doses and cytotoxicity at high doses. Table 1 shows the effect of various oligonucleotide (Neo1WT) and lipid concentrations on ESC targeting.…”

Section: Correction Efficiency Depends On Transfection Procedures Andmentioning

confidence: 99%

“…Within a single lipofection, the distribution of ssODN per cell is quite broad, with some cells taking up more than 100-fold more ssODN than others. 21 To determine if higher oligonucleotide uptake correlated with higher correction, we sorted ESC based on fluorescent oligonucleotide uptake. We transfected four dishes of pNeoS1Pur (clone 9) cells with a mixture of 80% Neo1WT and 20% Neo1WT-Cy5.…”

Section: Correction Efficiency Depends On Transfection Procedures Andmentioning

confidence: 99%

Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting

et al. 2006

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Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are o10 À4 , leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the basespecificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.

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Transfection by Cationic Liposomes Using Simultaneous Single Cell Measurements of Plasmid Delivery and Transgene Expression

Cited by 170 publications

References 26 publications

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

Gene delivery through cell culture substrate adsorbed DNA complexes

Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting

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