Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

<div><p>Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 10³ cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.</p></div

Fibroblast, SCN neural cells and HEK293 cells transfected Venus plasmid by W/O droplet electroporation.

<p>(A) Bright field, fluorescence and merge images of fibroblast cells from a subject aged 81 years old one day after electroporation. (B) 7 days after W/O droplet electroporation. (C) Bright field, fluorescence and merge images of SCN neural cells three days after W/O droplet electroporation. SCN cells had been differentiated to neural cells by incubation at higher culture temperature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144254#pone.0144254.ref026" target="_blank">26</a>]. (D) Twelve days after W/O droplet electroporation. All scale bars (A-D), 30 μm. (E) Bright field, fluorescence and merge images of HEK293 cells colonies were picked up and cultured for one month after W/O droplet electroporation. (F) Bright field, fluorescence and merge images of HEK293 cells colonies were cultured though frozen stock for a total of more than two months after W/O droplet electroporation. scale bars (D-E), 100 μm.</p

Image of the parallel W/O droplet electroporation electrode for the 8-well string of disposable 96-well plates.

<p>(A) Conductive electrodes were produced and set on an 8-well string in 96-well microwell plates for W/O droplet electroporation. (B), (C) Venus plasmid was transfected into some HEK293 cells using 8-well W/O droplet electroporation electrodes (Nepa Gene) for 96-well plates at 1.8–2.2 kV. Venus fluorescence signals were observed after incubation in 5 of 8 wells for 4 and 7 days following electroporation. Scale bars, 20 μm.</p

The droplet actuation device and cells transfected by the W/O droplet electroporation method.

<p>(A), (B) Two pieces of conductive tape were set parallel on a single cuvette or 8 wells in a line for 96-well plastic microwell plates. The droplet continued to bounce between the edges of the two electrodes. One was the ground electrode, and the other was the high-voltage electrode. Images of droplets bouncing between the anode and cathode in each well are shown. Many cells in droplets can be transfected simultaneously. (C) The upper row : Bright-field (BF), fluorescence, and merge images of HEK293 cells 24 hours after transfection by W/O droplet electroporation. Cells were examined at 24 hours after transfection to evaluate the expression of fluorescent protein (Venus). Scale bars, 100 μm. The lower row : BF, fluorescence, and merge images of HEK293 cells 24 hours after transfection by lipofection. (D) Variation of cell viability with time of droplet actuation determined by trypan blue staining. All experiments were performed at least twice.</p

Double and stable transfected cells by W/O droplet electroporation.

<p>(A), (B) Transgene plasmid DNAs of two kinds of fluorescent protein: green fluorescent protein (GFP) and Tag-RFP (red fluorescent protein), were successfully double transfected into HEK293 cells by W/O droplet electroporation for 3 minutes. Bright-field image and fluorescent GFP or RFP image of HEK293 cells (A) 5 days (71% of cells maintained GFP and 71% of cells maintained TagRFP signal) and (B) 8 days (41% of cells maintained GFP and 37% of cells maintained TagRFP signal) after W/O droplet electroporation. Scale bars, 30 μm. (C) Transgene plasmid DNAs of Venus and mCherry (red FP) were successfully double transfected into hippocampus primary neural cell lines by W/O droplet electroporation for 5 minutes. Scale bars, 30 μm. (D), (E) Bright-field image and fluorescent image of Venus and mCherry expression in human fibroblast cells (D) 2 days and (E) 10 days after W/O droplet electroporation. Scale bars, 30 μm.</p

Preparation of Chiral Bromomethylenecyclopropane and Its Use in Suzuki–Miyaura Coupling: Synthesis of the Arylmethyl‑(<i>Z</i>)‑cyclopropane Structure Core

A preparative method for an optically active bromomethylenecyclopropane unit and its practical conversion to (<i>Z</i>)-cyclopropane-containing chiral compounds via Suzuki–Miyaura coupling were established

Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state

<p>Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.</p

Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, −0.045, and −0.095 for λDNA and 0, −0.047, and −0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of −0.095 for λDNA and −0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of −0.045 and −0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA