Gene Electrotransfer

Kandušer, Maša; Pavlin, Mojca

doi:10.1016/b978-0-12-396533-2.00001-x

Cited by 5 publications

(2 citation statements)

References 67 publications

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“…Electrophoretic driving force causes siRNA electrophoretic displacement ( L ) during pulse application and accumulation at the cell membrane, which can be calculated similarly as published previously [Kandušer and Pavlin, ; Pavlin and Kandušer, ]:

L = μ E t_{E},

where t E is total pulse duration, and μ is mobility of siRNA molecule, defined as:

μ = \frac{e_{e f f}}{6 π η R_{g}},

where η is viscosity of water ( η H 2O = 9.5 × 10 −4 kgm −1 s −1 ) and R g is radius of gyration. DNA and RNA molecules behave as polymer chains, where the radius of gyration for a 22 bp long dsRNA is R g ≅ 1.8 nm [Viovy, ].…”

Section: Methodsmentioning

confidence: 98%

“…Electrotransfection efficiency in vitro and in vivo depends on parameters of electric pulses like electric field strength ( E ), pulse duration ( t ), number of pulses, and repetition frequency [Wolf et al, ; Mir et al, ; Rols and Teissié, ; Gehl et al, ; Golzio et al, ; Kandušer et al, ; Faurie et al, ; Hojman, ; Pavlin et al, ; Haberl et al, ; Pavlin and Kandušer, ]. Therefore, pulsing protocol needs to be optimized for the chosen application and used cell type [Kandušer et al, ; Pavlin et al, ; Kandušer and Pavlin, ]. Several aspects are important for efficient electrotransfection of siRNA: (i) electropermeabilization of plasma membrane; (ii) electrotransfer across the membrane into cytoplasm; and (iii) cell survival after electric pulse application [Paganin‐Gioanni et al, ].…”

Section: Introductionmentioning

confidence: 99%

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siRNA delivery into cultured primary human myoblasts – optimization of electroporation parameters and theoretical analysis

Lojk

Miš

Pirkmajer

et al. 2015

Bioelectromagnetics

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Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.

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L = μ E t_{E},

where t E is total pulse duration, and μ is mobility of siRNA molecule, defined as:

μ = \frac{e_{e f f}}{6 π η R_{g}},

Section: Methodsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

siRNA delivery into cultured primary human myoblasts – optimization of electroporation parameters and theoretical analysis

Lojk

Miš

Pirkmajer

et al. 2015

Bioelectromagnetics

Self Cite

View full text Add to dashboard Cite

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Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level

Haberl

Kandušer

Flisar

et al. 2013

The Journal of Gene Medicine

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Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA-membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol.

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New Insights into the Mechanisms of Gene Electrotransfer – Experimental and Theoretical Analysis

Pavlin

Kandušer

2015

Sci Rep

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Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol, and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (NDNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stable electropores or enters by electo-stimulated endocytosis, possibly dependent on pulse parameters. Understanding of the mechanisms enables the selection of optimal electric protocols for specific applications.

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Gene Electrotransfer

Cited by 5 publications

References 67 publications

siRNA delivery into cultured primary human myoblasts – optimization of electroporation parameters and theoretical analysis

siRNA delivery into cultured primary human myoblasts – optimization of electroporation parameters and theoretical analysis

Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level

New Insights into the Mechanisms of Gene Electrotransfer – Experimental and Theoretical Analysis

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