Improved Intracellular Delivery of Oligonucleotides by Square Wave Electroporation

Liu, Yueqin; Bergan, Raymond C.

doi:10.1089/108729001750072083

Cited by 20 publications

(5 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our observation is consistent with the nuclear localization of ODNs that was previously reported by using a standard electroporation approach [32].…”

Section: Figuresupporting

confidence: 93%

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

et al. 2008

View full text Add to dashboard Cite

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.

show abstract

“…Our observation is consistent with the nuclear localization of ODNs that was previously reported by using a standard electroporation approach [32].…”

Section: Figuresupporting

confidence: 93%

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Square wave electroporation is characterized by the voltage delivered, the duration of the pulse, the number of pulses and the length of the interval between pulses. This type of electroporation pulse allows the delivery of a more defined and regulated electrical pulse and has been associated with high transformation efficiencies in a variety of systems [ 27 ], including fungi [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

Transient transformation of Podosphaera xanthii by electroporation of conidia

et al. 2015

View full text Add to dashboard Cite

BackgroundPowdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.ResultsTo introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.ConclusionThis is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.

show abstract

“…Two types of pulses can be applied, the exponential decay which decreases during time and square waves, which allows a controlled pulse of fixed amplitude for a fixed period of time. In these experiments transfection efficiency, but not cell viability, was enhanced, using square waves, as has been shown in other studies as well (Liu and Bergan 2001). One would also like to apply sufficiently high enough voltage to permeabilize cells without a major cell loss.…”

Section: Discussionmentioning

confidence: 59%

Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line

Fyrberg

Lotfi

2010

Cytotechnology

View full text Add to dashboard Cite

In order to study nucleoside analog activation in the CEM cell line, a transfection protocol had to be optimized in order to silence an enzyme involved in nucleoside analog activation. Hematopoetic cell lines can be difficult to transfect with traditional lipid-based transfection, so the electroporation technique was used. Field strength, pulse length, temperature, electroporation media, siRNA concentration, among other conditions were tested in order to obtain approximately 70-80% mRNA and enzyme activity downregulation of the cytosolic enzyme deoxycytidine kinase (dCK), necessary for nucleoside analog activation. Downregulation was assessed at mRNA and enzyme activity levels. After optimizing the protocol, a microarray analysis was performed in order to investigate whether the downregulation was specific. Additionally two genes were differentially expressed besides the downregulation of dCK. These were however of unknown function. The leakage of intracellular nucleotides was also addressed in the electroporated cells since it can affect the DNA repair mechansism and the efficiency of nucleoside analogs. Three of these pools were increased compared to untreated, unelectroporated cells. The siRNA transfected cells with reduced dCK expression and activity showed reduced sensitivity to several nucleoside analogs as expected. The multidrug resistance to other drugs, as seen in nucleoside analog-induced resistant cells, was not seen with this model.

show abstract

Improved Intracellular Delivery of Oligonucleotides by Square Wave Electroporation

Cited by 20 publications

References 22 publications

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

Transient transformation of Podosphaera xanthii by electroporation of conidia

Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line

Contact Info

Product

Resources

About