Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.

Fechheimer, Marcus; Boylan, John F.; Parker, Sandra K.; Sisken, Jesse E.; Patel, Gordhan L.; Zimmer, Stephen G.

doi:10.1073/pnas.84.23.8463

Cited by 292 publications

(151 citation statements)

References 27 publications

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“…16 Similarly, lithotripter shock waves, a special form of ultrasound waves with high pressure amplitudes, have been shown to enhance the transfer of ribosome inactivating proteins into malignant cells in vivo. 17 Other in vitro reports indicate that genes can be transferred into mammalian cells 18 and plants 19 by ultrasound cleaning and sterilization equipment. In recent in vitro experiments sinusoidal ultrasound [20][21][22] and lithotripter shock waves were used to mediate plasmid DNA transfer into mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound

2000

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Gene therapy as a form of molecular medicine is expected to have a major impact on medical treatments in the future. However, the clinical use of gene therapy today is hampered by inadequate gene delivering systems to ensure sufficient, accurate and safe DNA uptake in the target cells in vivo. Nonviral transfection methods might have the advantage of safe application, but it would be helpful to increase their transfection rates, especially in vivo. In this study, we show that focused ultrasound provides an enhanced transfer of DNA plasmids in vitro and in vivo. In vitro, the ␤-galactosidase and luciferase DNA reporter plasmid were transfected into four cell lines (NIH 3T3 fibroblasts, malignant melanoma Mewo, HeLa, Dunning prostate tumor R3327-AT1). Ultrasound induced a 55-(Mewo) to 220-fold (AT1) stimulation resulting in transfection efficiencies in vitro

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Section: Introductionmentioning

confidence: 99%

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound

2000

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“…[17][18][19][20][21] Ultrasound treatment has been used to introduce DNA vectors into yeast, plant cells, and mammalian cells in vitro. [19][20][21] In this study, we have demonstrated the application of ultrasound treatment for gene transfer to primary tumors in mice. More than a two-log increase in systemic gene transfer to primary tumor by sonoporation without any effect on gene expression in nontumor tissue demonstrates the usefulness of ultrasound for systemic gene targeting to primary tumors.…”

Section: Discussionmentioning

confidence: 99%

“…Ultrasound-enhanced delivery to cells has been demonstrated in vitro by uptake of extracellular fluid, drugs, and DNA into cells. [17][18][19][20][21] In the present study we describe the combination of an ultrasound method and systemic administration of cationic lipid/DNA complexes that provides for specific enhancement in gene transfer to primary tumors. The enhancement in gene transfer was restricted to sonoporated tumors and no effects were observed in non-sonoporated tissues.…”

Section: Introductionmentioning

confidence: 99%

Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration

et al. 2000

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“…Inset: Observed resistance to anti-BPDE was proportional to the percentage of GST n + cells in the electroporated population. 11. Survival of reverted GST n+ COS cells after exposure to anti-BPDE in a colony-forming assay.…”

Section: Gst M-conferred Resistance To Anti-bpde Inmentioning

confidence: 99%

Recombinant glutathione S‐transferase (GST) expressing cells purified by flow cytometry on the basis of a GST‐catalyzed intracellular conjugation of glutathione to monochlorobimane

et al. 1991

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COS cells transiently expressing glutathione S-transferase (GST) m, Ya, or Yb, (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzoblpyrene (*)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 p M mClB for 3035 s during transit before being analyzed for fluorescence intensity and sorted. The apparent K , for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 pM. Stained GST Ya+ or Yb,+ cells catalyzed the conjugation 2 or 5 times more effectively than GST n+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 -+ 0.3-fold greater than that of the control (80 f 4 nmoYmin/mg protein). Upon a 5-fold purification of GST n+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P <0.001).Key terms: Resistance to carcinogens, reversion of transient expression, COS cells Glutathione S-transferases (GST,3 EC 2.5.1.18) are a family of isozymes that can catalyze the covalent addition of the tripeptide glutathione, present at 0.5-10 mM in mammalian cells (35), to a structurally diverse array of physiological and xenobiotic electrophiles (21,25,32,37,41,52,53). GSTs are ubiquitous in the animal and plant kingdoms (53), and Mannervik (32) has classified the mammalian cytosolic GSTs into three classes (Pi, Alpha, and Mu) based on structural, immunological, and enzymatic properties. Mammalian cytosolic GSTs constitute 1-10% of total cytosolic protein (21) and are composed of two subunits (23-28 kilodaltons each) (32) that dimerize by noncovalent interactions (51). As demonstrated with in vitro kinetic analyses, GSTs can conjugate glutathione to anticancer alkylating agents (2,3,9,49,59) [1606][1607][1608][1609][1610][1611][1612] 1991) and Ublacker et al. (Cancer Res 51: 1783-1788, 1991, based upon in vitro kinetic analyses, support our findings done in whole cells which show that the rat )I and a class GSTs catalyze the conjugation of GSH to monochlorobimane more efficiently than 71 class GSTs.

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Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.

Abstract: Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection ofmammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418

Cited by 292 publications

References 27 publications

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound

Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration

Recombinant glutathione S‐transferase (GST) expressing cells purified by flow cytometry on the basis of a GST‐catalyzed intracellular conjugation of glutathione to monochlorobimane

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