Abstract: The present invention relates to a method of introducing nucleic acids into cells by electroporation, comprising the step (A) of loading nucleic acids to the surface of an electrode; the step (B) of adhering cells on the obtained nucleic acid-loaded electrode surface; and the step (C) of applying electric pulses to the adhering cells. According to this method, not only efficient introduction of a gene into cells but also gene introduction at desirable timing and at desirable sites can be performed without damaging the adhering cells.

Abstract: The invention relates to methods of altering gene expression by homologous recombination in a multipotent adult progenitor cell (MAPC). In particular, methods of producing a recombinant MAPC, of correcting a genetic defect in a mammal, of providing a functional and/or therapeutic protein to a mammal, and of transforming a MAPC are provided. MAPCs containing an erogenous DNA as well as recombinant MAPCs and their differentiated progeny are also provided.

Abstract: Disclosed herein are a cloned canine and a production method thereof. The method comprises the steps of enucleating the oocyte of a canine to prepare an enucleated recipient oocyte, conducting nuclear transfer into the enucleated oocyte using a canine somatic cell as a nuclear donor cell under optimized conditions so as to prepare a nuclear transfer embryo, and transferring the nuclear transfer embryo into the oviduct of a surrogate mother. The present invention provides a method for producing cloned canines and thus, can contribute to the development of studies in veterinary medicine, anthropology and medical science such as the propagation of superior canines, the conservation of rare or nearly extinct canines, xenotransplantation and disease animal models.

Abstract: The present invention provides a medicine, comprising (a) an Otx2 protein or its partial peptide, or a salt thereof, or (b) a DNA or an RNA encoding an Otx2 protein or its partial peptide. The present medicine is useful as an agent for preventing, treating or suppressing progression of a retinal disease including retinal degeneration. In addition, the present medicine is useful, for example, as an agent for inducing differentiation from a retinal stem cell into a retinal photoreceptor cell, in the transplantation of a cell into the retina of patients suffering from retinal diseases.

Abstract: The present invention provides adjuvants, vaccines and therapies in which antigen presentation is enhanced through inhibition of negative immune regulators. The compositions and methods are useful for generating immune responses against antigens, including microbial pathogens and tumor-associated antigens, by way of inhibiting a negative immune regulator in a cell and providing a proinflammatory stimulus. In particular, nucleotides encoding inhibitors of negative immune regulators, antigens, and co-stimulatory molecules are contacted with immune cells in order to elicit a therapeutic or prophylactic response.

Abstract: The present invention provides a method and apparatus for transferring an agent into a cell. The method includes the steps of providing an agent outside of a cell and generating a vapor bubble and a plasma discharge between an avalanche electrode and a conductive fluid surrounding the cell. The vapor bubble and plasma discharge generate a mechanical stress wave and an electric field, respectively. The combination of this mechanical stress wave and electric field results in permeabilization of the cell, which in turn results in transfer of the agent into the cell.

Abstract: With the current invention, we provide new methods of enhancing the T-cell stimulatory capacity of human dendritic cells (DCs) and their use in cancer vaccination. The method comprises the introduction of different molecular adjuvants to human DCs through transfection with at least two mRNA or DNA molecules encoding markers selected from the group of: CD40L, CD70, constitutively active TLR4 (caTLR4), IL-12p70, EL-selectin, CCR7 and/or 4-1 BBL; or in combination with inhibition of SOCS, A20, PD-L1 and/or STAT3 expression, for example through siRNA transfection. We could show a clear increase in the immunostimulatory capacity of DCs obtained in this way, enabling them to elicit an unexpectedly high T-cell immune response in vitro.

Abstract: A pulse generator circuit may include a diode configured to operate as an opening switch, a tank circuit in series with the diode having an admittance that is switchable from a first value to a second value that is different from the first value, and a switching system configured to cause the tank circuit to switch between the first value and the second value. The diode may saturate in less than 100 nanoseconds. A saturable core transformer may operate as a switch that controls the opening of the diode. The pulse generator may generate a plurality of pulses, each having a length of no more than 3 nanoseconds and an amplitude of at least 1 kilovolt. Electrodes may be connected to the pulse generator to deliver the plurality of pulses to biological cells.

Abstract: The invention relates to a method for treating biomaterial using at least one electrical field generated by a first voltage pulse which is terminated once the value for an electrical parameter has exceeded or dropped below a preset limit. After the first voltage pulse has been terminated, it is continued by an additional voltage pulse. The invention also relates to a circuit arrangement comprising at least one storage device for electrical charges to generate at least one voltage pulse by selectively discharging the storage device, and at least one control unit for controlling the discharge. The present invention provides a controller for monitoring the chronological progression of the voltage pulse. This controller controls at least one continuation of discharge after termination.

Abstract: Antisense sequences, including duplex RNAi compositions, which possess improved properties over those taught in the prior art are disclosed. The invention provides optimized antisense oligomer compositions and method for making and using the both in in vitro systems and therapeutically. The invention also provides methods of making and using the improved antisense oligomer compositions.

Abstract: A method for facilitating a delivery of a molecule into an interior space of a cell includes the steps of introducing a molecule into a target tissue comprising a cell and applying a substantially continuous low-level electric field to the target tissue. The field is applied for a duration sufficient to effect a change in porosity the cell of the target tissue sufficient to facilitate an entry of a desired molecule into an interior of the cell.

Abstract: Disclosed is a device for processing biological material which at least comprises a chamber at least closable to the outside and having an inner space for receiving the biological material. The chamber comprises at least one electrode placed in contact with the inner space for generating an electric field. Also disclosed is a method for processing biological material. The biological material is introduced into the inner space above and the electrode can generate an electric field after said biological material is introduced by applying voltage to said electrode and a further electrode in contact with the inner space. The chamber comprises at least one inlet line having at least one opening arranged close to the electrode. The biological material is almost completely rinsed out of the inner space after the electric field is generated, via a solution guided via an inlet line of the chamber along at least one electrode.

Abstract: In method of injecting a substance into a living cell having a cell membrane, the substance, the cell and a liquid are placed into a tapering passage. Energy is applied to the cell, thereby inducing poration. To sort cells, a cellular suspension is placed in a tapering passage, including a narrow end that defines an opening that has a dimension corresponding to a cell size. An acoustic wave is applied, thereby forcing cells having a cell size smaller than the selected cell size through the opening, with a portion of the cells having a cell size not smaller than the selected cell size not forced through the opening. To extract material from a cell, an electric field and an acoustic wave are applied, thereby causing the cell membrane to allow the material to pass out of the cell.

Abstract: Disclosed is a method for treating biological material via electrical current, in which the biological material is added to a small volume of a buffer solution having relative high ionic strength. A strong electrical field is generated in the buffer solution by a high voltage pulse having a preset duration. The biological material is added to at most 50 ?l of a buffer solution with an ionic strength of at least 100 mmol/l. By at least one voltage pulse having a preset duration of at least 10 ?s, an electrical field with a field strength of at least 1 kV/cm is generated in the buffer solution. The voltage pulse is hereby interrupted at least once for a duration of at least 100 ?s and is then again continued.

Abstract: Transfecting biology cells with nucleic acid molecules (DNA, siRNA) is an essential prerequisite in elucidating how genes function in complex cellular context and how their activities could be modulated for therapeutic intervention. Traditionally studies are carried out on a low throughput gene-by-gene scale, which has created a huge bottleneck in functional genomic study and drug discovery. Development of high-throughput cell transfection technology will permit functional analysis of massive number of genes and how their activities could be modulated by chemical or biological entities inside cells. This invention describes design, construction of device and apparatus for high throughput effective cell transfection. Procedures and protocols for using the device and apparatus are also described in the application. Novel methods of using the device in cell-based assays are also disclosed.

Abstract: Methods and composition of induction of pluripotent stem cells are disclosed. For example, in certain aspects methods for generating induced pluripotent stem cells using reporter genes are described. Furthermore, the invention provides novel reprogramming vectors employing reporter genes.

Abstract: Electro-poration device for the permeabilization of cells contained in a substrate comprising a signal generator for generating a stimulating signal applied by means of electrodes to the substrate wherein an electric field permeabilizing the cells membranes is induced. The device calculates and monitors the instantaneous value or the ratio GT between the current flowing through the substrate and the voltage of the stimulating signal applied to the substrate; in particular, the stimulating signal is applied in a modified manner according to the shape of an initial portion of the waveform of a curve CGT representing the value of ratio GT in successive instants after the application of the stimulating signal. The system will achieve permeabilization of cells in substrate with no or minimum damage, allowing to introduce or to extract molecules in or from the electropermeabilized living cells.

Abstract: The invention provides improved methods of producing dendritic cells (“DCs”) that transiently express a membrane homing peptide and optionally at least one additional antigen. These DCs have the ability to home to lymph nodes in vivo. In some embodiments, these DCs can be administered to a patient intravenously and can subsequently home to lymph nodes and stimulate an immune response. The methods and DCs of the invention are useful for the treatment of various diseases and disorders.

Abstract: Adherent cells on the surface of a disk are transfected by electroporation between coaxial circular cylinders with electrodes on the opposing surfaces on either side of the annular space between the cylinders by placing a buffer solution containing the transfecting species in the annular space over the cell and applying an electric potential between the electrodes.

Abstract: Vectors and methods for the production of influenza viruses suitable as recombinant influenza vaccines in cell culture are provided. Bi-directional expression vectors for use in a multi-plasmid influenza virus expression system are provided. Additionally, the invention provides methods of producing influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells (e.g., Vero and/or MDCK) and further provides influenza viruses with enhanced replication characteristics. In addition, the present invention includes an improved method of rescue, wherein animal cells (e.g., SF Vero cells) are electroporated with plasmids and vectors of the invention.

Abstract: The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage.

Abstract: The present invention provides methods and compositions for reprogramming somatic cells to a more primitive state, such as induced pluripotent stem cells, using homologous recombination. The induced pluripotent stem cells generated by the methods of the present invention are useful in a variety of therapeutic applications in the treatment and prevention of diseases and disorders.

Abstract: The present invention provides ungulates, including pigs, expressing CTLA4-Ig, as well as tissue, organs, cells and cell lines derived from such animals. Such animals, tissues, organs and cells can be used in research and medical therapy, including xenotransplanation. In addition, methods are provided to prepare organs, tissues and cells expressing the CTLA4-Ig for use in xenotransplantation, and nucleic acid constructs and vectors useful therein.

Abstract: Disclosed are methods and compositions to genetically modify substantially intact cells having cosmetic function to enhance the cosmetic appearance in mammals so as to enhance and/or maintain a biochemical and/or physiological process that has a positive effect on cosmetic appearance. The methods and compositions may provide cosmetic benefits such as reduced skin sagging, increased skin thickness, reduced wrinkles, increased skin thickness and collagen content, increased skin tone and elasticity, increased skin hydration, and improved skin texture and color.

Abstract: The invention relates to methods for altering the fate or differentiation status of somatic cells by RNA transfer. These methods can be used to transdifferentiate or dedifferentiate somatic cells of one phenotype or lineage into pluripotent cells or into somatic cells of a different lineage or phenotype.

Abstract: A method of producing propagation-defective Vesicular Stomatitis Virus (VSV) in a cell culture is provided. The method involves introducing a plasmid vector encoding an optimized VSV G gene into a cell; expressing VSV G protein from the optimized VSV G gene; and introducing a propagation-defective VSV into the cell expressing the VSV G protein encoded by the optimized VSV G gene. The method further includes growing the cells in culture; and recovering the propagation-defective VSV from the culture.

Abstract: The present invention provides an improved method for gene delivery in eukaryotic cells by electroporation, preferably in human hematopoietic cells, particular dendritic cells. The method of the invention is superior to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for gene delivery, including tumor antigen loading of dendritic cells.

Abstract: A method for the production and use of multipotential stem-like cells is disclosed. The preparation utilized in this method is characterized by the contact of low level electrical currents with cultures of fibroblasts or other -blast cells enriched by fibroblast growth factor and other nutrients. The electrical current is conducted by means of silver electrode(s) brought into contact with the fibroblast preparation or other -blast cell preparation cultured for that purpose. The cells of the preparation may be used in applications that require the use of stem cells, including therapeutic applications, without the need for human fetuses or human umbilical cords or penetrating human bones to extract bone marrow. The cells thus produced have the ability to redifferentiate into endoderm, ectoderm and mesoderm to form any tissue of the body except the lens of the eye. Any cell found in the blood may be copied and multiplied.

Abstract: The electroporation array is comprised of three technologies: microwire glass electrodes, microelectronic multiplexer stimulator chips and microfluidic flow chamber. Various substances, such as genes, gene silencing RNAi, gene inhibition agents or drugs, can be perfused into the microfluidic flow chamber. The entry of the various substances into the cells will be facilitated by electroporation. An applied electric potential causes nanoscale pores to open in the cell membrane allowing substances in the solution to freely diffuse into the cell. The specific cells selected for electroporation are defined using the computer controlled microelectronic stimulator array. An “image” of which electrodes within the array to apply the electric potential to, and thus electroporate, is de-multiplexed onto the array. All the selected electrodes deliver a current pulse varied by the intensity of the de-multiplexed “image”.

Abstract: A process is provided for the intracellular manipulation of a biological cell (3) which is positioned adhering to a support area (5) in a culture medium (2). Inside the support area (5) for the cell (3) an opening into the membrane of the cell (3) is created spaced from its support edge. The edge of the cell membrane surrounding the opening, adhering to the support area (5), thus seals off the cell fluid situated in the interior of the cell (3) from the culture medium (2) and insulates the cell fluid against the culture medium (2). The interior of the cell (3) is manipulated through the opening. An apparatus for implementing the process is also provided, including an object carrier (4) with a support area (5) for adhering the cell and a poration tool (6) for creating the opening in the cell membrane. The poration tool (6) may be any of various chemical, mechanical and/or electrical devices.

Abstract: Chamber for treating cells contained in a suspension in the electrical field with a beaker made from electrically non-conductive material, into which an elongate core made from electrically non-conductive material is at least partially inserted axially through an opening, between the beaker and the core a gap being present to receive the suspension; at least two electrodes made from electrically conductive material arranged on the outer face of the core facing the gap, between which an electrical field can be created to treat cells in a suspension contained in the gap by applying a voltage; the thermal expansion coefficient of the material of the electrodes and of the material of the core being matched with one another such that the electrodes do not substantially alter their position relative to the core in the temperature range from ambient temperature to temperatures reached during autoclaving and/or sterilising.

Abstract: Described herein are platforms and consumables for performing gene/siRNA/protein/peptide delivery screens in high throughput, including an automation-compatible transfection methodology that can be used to gain entry of oligonucleotides, proteins, peptides and other non-permeable molecules (e.g., some small organic compounds) into cells. Electrical stimulation is provided to cells, e.g., neurons or myocytes, in culture through application of pulsed fields directly to the cells in culture. The plate design is used in conjunction with a custom-designed circuit that allows the user to select pulse type, duration, etc.

Abstract: A nucleic acid formulation for use in gene delivery comprising a nucleic acid and an anionic polymer is disclosed. Examples of the anionic polymer includes anionic amino acid polymer or poly-amino acid (such as poly-L-glutamic acid, poly-D-glutamic acid, poly-L-aspartic acid, poly-D-aspartic acid), poly-acrylic acid, polynucleotides, poly galacturonic acid, and poly vinyl sulfate.

Abstract: The invention provides hollow-tip-electrodes for spatially localized delivery of substances to one or more biological targets present in a population comprising target and non-target molecules, macromolecules, and/or cells. The invention also provides electrode plates for receiving one or more of such tips, tip-electrode plates comprising electrode plates comprising one or more electrode tips, and systems comprising tip-electrodes and containers for containing one or more biological targets, e.g., such as molecules, macromolecules, and/or cells. The invention further provides methods for using such systems and components thereof. In one preferred aspect, the systems are used for spatially confined electroporation of cells and cell structures. The invention facilitates high throughput screening of agents (e.g., such as drugs) that act on intracellular targets.

Abstract: A system and method are described for electroporating a sample that utilizes one or more sets of electrodes that are spaced apart in order to hold a surface tension constrained sample between the electrodes. The first electrode is connected to the lower body of the system while the second electrode is connected to the upper body. Both electrodes are connected to a pulse generator. Each electrode has a sample contact surface such that the first electrode and the second electrode may be positioned to hold a surface tension constrained sample between the two sample contact surfaces and the sample may receive a selected electric pulse.

Abstract: Electroporation of mammalian cells is performed to high efficiency by use of a buffer solution containing trehalose, sucrose, or both, in addition to an inorganic phosphate buffer, an organic sulfonic acid buffer, a halide salt of an alkali or alkaline earth metal, a chelating agent, an alkyl methyl sulfoxide, and a nucleotide triphosphate, with substantially no sodium. This buffer solution is effective both in the electroporation of cells that are suspended in the buffer solution and in the electroporation of cells that are fixed on a surface.

Abstract: Described is a method for method for transferring material through the membrane of at least one cell, wherein the transfer is carried out in the presence of trehalose. This method is applicable in particular in the field of genetic engineering and biotechnology.

Abstract: The intramuscular electroporated injection of a protease-resistant growth hormone-releasing hormone (“GHRH”) cDNA into rat dams at 16 days of gestation resulted in the enhanced long-term growth of the FI offspring. The offspring were significantly heavier by one week of age and the difference was sustained to 10 weeks of age. Consistent with their augmented growth, plasma IGF-I concentration of the FI progeny was increased significantly. The pituitary gland of the offspring was significantly heavier, and contained an increased number of somatotropes (cells producing GH) and lactotrophs (prolactin-secreting cells), and is indicative of an alteration in cell lineages. These unique findings demonstrate that enhanced GHRH expression in pregnant dams can result in intergenerational growth promotion, by altering development of the pituitary gland in the offspring.

Abstract: The present invention provides a method for producing large quantities of transmembrane and cytosolic proteins in a mammalian muscle cell. The method involves transfecting skeletal muscle cells in vivo with nucleic acids encoding the proteins by electroporation. The invention results in production of the desired protein on the order of about 2-3 magnitudes more as compared to standard methods, allowing for various biological uses including purification and crystallization.

Abstract: This disclosure features vectors, targeting vectors, modified artificial chromosomes, herpes virus particles in which such chromosomes are packaged, and methods of using the herpes virus particles to identify therapeutic agents and targets for therapeutic intervention.

Abstract: A system and method are described for electroporating a sample that utilizes one or more sets of electrodes that are spaced apart in order to hold a surface tension constrained sample between the electrodes. The first electrode is connected to the lower body of the system while the second electrode is connected to the upper body. Both electrodes are connected to a pulse generator. Each electrode has a sample contact surface such that the first electrode and the second electrode may be positioned to hold a surface tension constrained sample between the two sample contact surfaces and the sample may receive a selected electric pulse.

Abstract: The present invention includes compositions and methods for transforming cells into glucose-responsive, insulin-production cells using a construct that expresses betacellulin and PDX1, e.g., transforming pancreatic acinar cells using one or more expression vectors that expressed betacellulin and PDX1 using ultrasound targeted microbubble destruction (UTMD).

Abstract: A buffer solution for suspending animal or human cells and for dissolving biologically active molecules in order to introduce the biologically active molecules into the cells using electric current. The buffer solution includes HEPES and at least 10 mmol*1?1 magnesium ions (Mg2+). The buffer solution has a buffer capacity of at least 20 mmol*1?1*pH?1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C. The buffer solution also has an ionic strength of at least 200 mmol*1?1.

Abstract: A buffer solution for suspending animal or human cells and for dissolving biologically active molecules in order to introduce said biologically active molecules into the cells using electric current. The buffer solution includes at least one of sodium succinate, mannitol and sodium lactobionate. The buffer solution has a buffer capacity of at least 20 mmol*l?1*pH?1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C., and an ionic strength of at least 200 mmol*l?1.

Abstract: Described are methods for electrical treatment of biological cells, in particular using electrical field pulses, involving the steps: arrangement of the cells (1) on apertures (2) of a solid planar carrier element (3) which divides a measuring chamber (10) into two compartments (11, 12); and temporary formation of an electrical treatment field which permeates the cells, wherein an alternating-current impedance measurement takes place on the carrier element (3), and from the result of the alternating-current impedance measurement, a degree of coverage of the carrier element and/or healing of the cells after electrical treatment are/is acquired. Also described are devices for implementing the methods.

Abstract: A molecule introducing apparatus is provided with a power supply circuit 20 for supplying power, a step-up transformer 30 which is supplied with power from the power supply circuit 20 and which outputs a high voltage, a switching transistor 22 that blocks off and allows the supply of power from the power supply circuit 20 to the step-up transformer 30 and which generates an instantaneous high voltage, and a pair of electrode probes 50 for applying the instantaneous high voltage generated at the step-up transformer 30 to a predetermined region in a body. The molecule introducing apparatus oscillates DNA arranged outside a cell by the instantaneous high voltage so as to introduce the DNA into the cell.

Abstract: The subject invention relates to a method for introducing an exogenous gene into a stem cell, which comprises introducing the exogenous gene into the stem cell under specific electroporation conditions. The invention also relates to a stem cell stably expressing an exogenous gene, a transgenic animal and a pharmaceutical composition.

Abstract: The invention relates to a buffer solution for suspending animal or human cells and for dissolving biologically active molecules in order to introduce biologically active molecules into cells using an electric current. The inventive buffer solution has a buffering capacity of at least 20 mmol?1×pH?1 and an ionic strength of at least 200 mmol×1?1 during a change to the pH value from pH 7 to pH 8 and at a temperature of 25° C. The use of a buffer solution of this type allows biologically active molecules to be introduced into animal and human cells with a high degree of transfection efficiency and at the same time a low cell mortality. Different cell types, in particular dormant and actively dividing cells of low activity, can be successfully transfected in the buffer solution.

Abstract: An artery wall binding peptide (AWBP) based on the artery wall cell-binding domain of apolipoprotein B-100 was conjugated to a cationic backbone configured for forming a complex with a nucleic acid to produce a composition that enhances gene transfer to artery wall cells. An illustrative cationic backbone is poly(ethylene glycol)-grafted-poly(L-lysine) (PEG-g-PLL). Methods of making and using the composition for gene transfer are also described.

Abstract: An expression vector capable of expressing high levels of heterologous proteins having a cytomegalovirus (CMV) enhancer 5? upstream from a myeloproliferative sarcoma virus (MPSV) promoter.