Abstract: A method for gene therapy using small fragment homologous replacement. The method introduces small fragments of exogenous DNA into regions of endogenous genomic DNA virtually homologous to the exogenous DNA. The exogenous DNA fragments contains sequence modification that correct mutations in the endogenous DNA or introduce mutations that alter cellular or an infecting pathogen phenotype.

Abstract: The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations in blood by electroporation to achieve therapeutically desirable changes in the physical characteristics of the various cell populations in blood.

Abstract: The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations in blood by electroporation to achieve therapeutically desirable changes in the physical characteristics of the various cell populations in blood.

Abstract: The present invention provides a composition of matter for introducing an exogenous nucleic acid molecule into a target cell, comprising a liposome, a ligand polymeric scaffold, wherein the ligand can bind to a cell surface receptor or molecule. The invention also provides methods for introducing an exogenous nucleic acid molecule into a target cell using the composition of matter.

Abstract: A method and device for delivering a quantity of at least one material to a selected single cell in a manner that permits impingement of material with the cell. The material travels at a suitable trajectory and velocity and is ejected from an electronically controllable fluid device.

Abstract: The invention relates to a method for permeabilization of a cell structure consisting of a single cell, an intracellular structure or an organelle comprising the following steps: (a) microelectrodes, preferably two carbon fiber electrodes or hollow fiber electrodes, are provided, (b) the microelectrodes are connected to a power supply, (c) the electrodes, individually controlled by high-graduation micromanipulators, are placed close to the cell structure at an appropriate inter-electrode distance, and (d) a highly focused electric field of a strength sufficient to obtain electroporation is applied between the electrodes. The method may be used in order to transfer cell impermeant solutes, such as drugs or genes, into the cell structure or out of the cell structure, in biosensors, in the treatment of tumours and neurodegenerative diseases and in the study of biophysical processes.

Abstract: The present invention is directed to an electrofusion microelectrode used in the alignment, manipulation, fusion, or electroporation of cells. This device is particularly useful for transplantation of cells and cellular components.

Abstract: Tn5 transposase (Tnp) mutants that have higher transposase activities than the wild-type Tnp are disclosed. The Tn5 Tnp mutants differ from the wild-type Tnp at amino acid positions 54, 242, and 372 and have greater avidity than the wild-type Tnp for at least one of a wild-type Tn5 outside end sequence as defined by SEQ ID NO:3 and a modified Tn5 outside end sequence as defined by SEQ ID NO:5. Also disclosed are various systems and methods of using the Tnp mutants for in vitro or in vivo transposition.

Abstract: Provided herein are methods for producing alphavirus replicon particles in high yield; replicon RNAs are electroporated into permissive cells, where the cells are at a relatively high density, together with at least one helper nucleic acid providing the necessary functions for packaging. After a growth period in appropriate medium, alphavirus replicon particles are harvested from the surfaces of the cells in which they were produced using a salt wash in which the salt concentration is from about 0.2 to about 5 M sodium chloride, calcium chloride, magnesium chloride, potassium chloride, ammonium acetate, ammonium bicarbonate, among others. After dilution, if necessary, the particles can be purified by a suitable chromatographic technique.

Abstract: Electroporation is performed in a controlled manner in individual and multiple biological cells present in biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general.

Abstract: The present invention provides compositions useful in and methods for producing populations of infectious, replication-defective alphavirus replicon particles that contain no replication-competent alphavirus particles, as determined by passage on cells in culture. The compositions include helper and replicon nucleic acid molecules that can further reduce the predicted frequency for formation of replication-competent virus and can optimize manufacturing strategies and costs.

Abstract: The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations in blood by electroporation to achieve therapeutically desirable changes in the physical characteristics of the various cell populations in blood.

Abstract: An apparatus and method are disclosed for electromanipulation of at least one cell or cell-like structure having cell-like membranes, the method comprising the steps: (a) at least one cell or cell-like structure is transported from one or more sample containers located on a chip through microchannel(s) located on said chip into a chamber located on said chip, wherein said chamber contains electrode(s) connected to a voltage generator, wherein said microchannel provides a fluid contact between the sample containers, (b) said cell or cell-like structure(s) is placed close to said at least one electrode, and (c) an electrical field is applied and focused on said cell or cell-like structure(s), said electrical field being of a strength sufficient to obtain pore-formation or fusion of said at least one cell or cell-like structure with another cell or cell-like structure(s) present in said chamber.

Abstract: The present invention is directed to a recombinant vector for transforming yeast and a process for transforming yeast thereby, more particularly to a recombinant vector comprising a gene encoding a mutated L41 protein having cycloheximide-resistant activity and a ribosomal DNA. The recombinant vector and the process for transforming thereby of the present invention is applicable to the efficient and stable integration of desired foreign DNA into yeast genome, thus providing useful tools for the production of a natural pigment, astaxanthin.

Abstract: The controllable electroporation system and method described herein allows control over the size, the number, the location, and the distribution of aqueous pores, thus increasing flexibility of use. The herein described system and method for controllable electroporation generally employs at least two actuating sub-systems and sub-processes. One sub-system and sub-process employs a relatively broad effect in order to weaken the membrane, a broad effect sub-system. Another sub-system and sub-process employs a relatively narrow effect in order to localize the position of the pore in the membrane, a narrow effect sub-system.

Abstract: The present invention provides a method and apparatus adapted to facilitate the entry of a preselected molecule into the intracellular space of a cellular sample through the use of ions generated by a corona charge source. With the present method and apparatus, molecules are manipulated within cells and in the extracellular space surrounding the cells. Manipulation enhances the permeability of cell barriers to allow the subsequent introduction of molecules of interest into the interior of a cell.

Abstract: An object of the invention is to provide a method of treating biological cells prior to subjecting the biological cells to cell fusion pulses which includes the step of treating the biological cells with pre-fusion electric field waveforms which change amplitude in a non-linear way with respect to time, such that the biological cells are first aligned with a relatively low amplitude, long duration pre-fusion electric field waveform and then compressed with a relatively high amplitude, short duration pre-fusion electric field waveform resulting in increased cell membrane contact prior to being subjected to cell fusion. The non-linear pre-fusion electric field waveforms can change in a stepped way, in a continuous way, in a sigmoidal way, with step-wise increasing waveforms in adjacent steps, with step-wise increasing waveforms in non-adjacent steps, and in accordance with non-linear algorithms.

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 is a composition for percutaneous administration suitable for electroporation. Percutaneous absorption of a drug and so forth in a composition for electroporation can be promoted by adding alkaline earth metal ions to the composition.

Abstract: This invention provides improved electroporation methods for transferring nucleic acids of interest into host cells, wherein the host cells are (1) suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative, (2) mixed with the nucleic acids of interest, and (3) electrically treated. Also, this invention provides for kits used in the method for transferring nucleic acids into host cells.

Abstract: The present invention is directed to isolated nucleic acid molecules that encode LIM mineralization protein, or LMP. The invention further provides vectors comprising nucleotide sequences that encode LMP, as well as host cells comprising those vectors. Moreover, the present invention relates to methods of inducing bone formation by transfecting osteogenic precursor cells with an isolated nucleic acid molecule comprising a nucleotide sequence encoding LIM mineralization protein. The transfection may occur ex vivo or in vivo by direct injection of virus or naked plasmid DNA. In a particular embodiment, the invention provides a method of fusing a spine by transfecting osteogenic precursor cells with an isolated nucleic acid molecule having a nucleotide sequence encoding LIM mineralization protein, admixing the transfected osteogenic precursor cells with a matrix and contacting the matrix with the spine.

Abstract: DNA is efficiently transformed into a host by electroporation in the presence of a methylation package, which greatly improves the efficiency of the transformation. The methylation package comprises a source of cysteine, such as cysteine, homocysteine, or glutathione, with optional iron and magnesium ions.

Abstract: A single cell electroporation assembly and method involve delivery of a substance into a single cell. The substance is combined into an electroporation fluid and placed into a container having a distal opening. The distal opening of the container is placed in proximity to a target cell. Electrical pulses are delivered between a first electrode which is at least partially disposed in the container, and a second electrode outside the cell, the cell being positioned between the distal opening and the second electrode. The electrical pulses induce temporary formation of pores in the cell membrane and the substance enters the cell through the pores by passive diffusion or by active electrophoretic motion.

Abstract: The invention provides primary and secondary cells that are transfected with a nucleic acid molecule that encodes erythropoietin, clonal or heterogenous strains of such cells, and methods of producing these cell strains.

Abstract: The present invention relates to a method for detection and interpretation of loss-of-function or gain-of-function mutations for test genes of interest. The genes of interest include those associated with inherited genetic disorders. The present invention involves the process of obtaining a sample of genetic material from an individual in the form of tissue or cells, separation of the genetic material from the cells of the individuals into haploid sets by transferring the individual chromosomal entities into a population of target cells, and monitoring the target cell population for successful transfer and expression of the test genes of interest using various functional, immunological and structural assays.

Abstract: The invention relates to a method for selectively releasing an agent loaded into a red blood cell, comprising electrosensitizing the red blood cell by application of an electric field and subsequently disrupting the cell selectively using ultrasound.

Abstract: A method is provided for introducing nucleic acid into a cell, by contacting the cell with a nucleic acid and applying a low electrical field impulse for a long pulse length. A method is provided for introducing a polypeptide into a cell, by contacting the cell with the polypeptide and applying a low electrical field impulse for a long pulse length.

Abstract: The invention features a method of delivering DNA to a spermatogonium by infusing DNA in situ into a testicle of a non-human animal and administering a condition or substance to the testicle to increase uptake of DNA by the spermatogonium

Abstract: This invention provides methods for producing and selecting host cells that better survive transformation treatment by subjecting host cells to conditions that alter them, subjecting the altered cells to transformation conditions, and selecting host cells that survive the transformation conditions. This invention also provides methods for transferring nucleic acids of interest into host cells, using cells that are better able to survive transformation treatment. Also, this invention provides kits for producing or selecting host cells in transformation treatments, as well as, kits comprising various host cells that may be utilized in transformation experiments.

Abstract: The invention provides compositions and methods for introducing bioactive agents into cells. Bioactive agents are provided together with a delivery vehicle and a cell is subjected to electroporation, thereby resulting in the introduction of the bioactive agent into the cell.

Abstract: Disclosed is a method for penetration of lipid bilayer membranes in order to insert at the tip of a hollow needle-shaped object, such as a micropipet-, into a container formed of a lipid bilayer membrane, wherein said container is placed between said needle-shaped object, with the tip of said needle-shaped object placed in contact with said conainer in such a way that it applies a mechanical force to the lipid membrane of said container, thus mechanically straining it, and a second electrode, whereupon a transient electric pulse of 1-to-103 V/cm is applied between the electrodes, resulting in a focused electrical field over said container C which induces a dielectric breakdown of the lipid bilayer causing the needle-shaped object to penetrate the container. Disclosed is also an electroinjection method based on the above method, wherein substances are introduced through the needle-shaped object and into the container after penetration of the needle-shaped object.

Abstract: The invention relates to a method for permeabilisation of a cell structure consisting of a single cell, an intracellular structure or an organelle comprising the following steps: (a) microelectrodes, preferably two carbon fibre electrodes or hollow fibre electrodes, are provided, (b) the microelectrodes are connected to a power supply, (c) the electrodes, individually controlled by high-graduation micromanipulators, are placed close to the cell structure at an appropriate inter-electrode distance, and (d) a highly focused electric field of a strength sufficient to obtain electroporation is applied between the electrodes. The method may be used in order to transfer cell impermeant solutes, such as drugs or genes, into the cell structure or out of the cell structure, in biosensors, in the treatment of tumours and neurodegenerative diseases and in the study of biophysical processes.

Abstract: The invention features a method of delivering DNA to a spermatogonium by infusing DNA in situ into a testicle of a non-human animal and administering a condition or substance to the testicle to increase uptake of DNA by the spermatogonium.

Abstract: The invention relates to a novel method allowing the transport of DNA and/or other biologically active molecules into the nucleus of eukaryotic cells using electrical current, independently from cell division and with low cell mortality.

Abstract: According to the invention, there is provided a vector for the expression of immunoglobulin-cytokine fusion proteins in malignant B cells at least containing operably linked to each other (a) a region of at least 1.5 kb which is homologous to a region of the &mgr; intron or the &kgr; intron and which lacks a functional C&mgr; or C&kgr; enhancer or contains a non-functional C82 or C&kgr; enhancer; (b) at least one DNA sequence encoding a domain of an immunoglobulin or a part thereof; (c) a DNA sequence encoding a cytokine; and (d) a marker gene selectable in eukaryotic B cells and lacking a functional enhancer region wherein the expression of said marker following integration is controlled by the cellular C&mgr; or C&kgr; enhancer.

Abstract: The present invention relates to transfected primary and secondary somatic cells of vertebrate origin, particularly mammalian origin, transfected with exogenous genetic material (DNA) that encodes erythropoietin or an insulinotropin (e.g., derivatives of glucagon-like peptide 1 (GLP-1)), methods by which primary and secondary cells are transfected to include exogenous genetic material encoding erythropoietin or an insulinotropin, methods of producing clonal cell strains or heterogenous cell strains that express erythropoietin or an insulinotropin, methods of gene therapy, in which the transfected primary or secondary cells are used, and methods of producing antibodies using the transfected primary or secondary cells.

Abstract: The present invention relates to a composition which is intended for transferring at least one therapeutically active substance into the interior of a target cell, characterized in that it comprises a mixture of at least one therapeutically active substance and at least one polar compound which is selected from a specific group of aprotic polar compounds. The therapeutically active substance is preferably a polynucleotide, and the polar compound is DPSO, which improves the ability of the polynucleotide to be transfected into the interior of the cells. The compositions according to the invention can be used as a diagnostic therapeutic, prophylactic or vaccinal medicament for treating the human or animal body by means of gene therapy.

Abstract: The invention relates to an apparatus for and method of introducing a substance into an object, particularly into a cell or cellular material. In a preferred arrangement apparatus receives the cell and preferably locates it between first and second electrodes and applies a voltage pulse to cause a disruption in the cell wall. This causes the cell to become permeable. The substance may then be introduced, for example under a fluid pressure. Cells may then be inspected and sorted into transfected and non-transfected types. This may be achieved automatically, for example by using electrophoresis.

Abstract: Disclosed herein is a novel cell line of human pancreatic cells that secrete insulin in a glucose-dependent manner. The cell line comprises pancreatic cells, such as PANC-1 cells, which are transfected so as to express IDX-1 and cultured in GLP-1. The cell line may be used to investigate the function and development of pancreatic cells, as well as to test the efficacy of drugs that stimulate insulin secretion.

Abstract: The present invention relates to a process for targeted replacement of at least a part of an endogenous gene by at least a part of a foreign gene or targeted insertion of at least a part of a foreign gene at a targeted site in an endogenous gene in a cell by homologous recombination.

Abstract: Expression cassettes for transforming Pichia ciferrii and their use are disclosed. The present invention relates to expression cassettes containing Pichia ciferrii ribosomal DNA fragment, CYHr gene resistant to cycloheximide, and a desired gene. Expression cassette further comprising Pichia ciferrii GAPDH promoter gene which allows an increase in the expression of the desired gene also provided. Moreover, the present invention provides a process for producing tetraacetyl phytosphingosine using transformed Pichia ciferrii cells with the expression cassettes in a higher yield.

Abstract: The present invention is directed toward a method of enhancing transfection efficiency by administering a nucleic acid to a salivary gland and electroporating the salivary gland.

Abstract: Electroporation is performed in a controlled manner in either individual or multiple biological cells or biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general. In addition, a particular method and apparatus are disclosed in which electroporation and/or mass transfer across a cell membrane are accomplished by securing a cell across an opening in a barrier between two chambers such that the cell closes the opening. The barrier is either electrically insulating, impermeable to the solute, or both, depending on whether pore formation, diffusive transport of the solute across the membrane, or both are sought.

Abstract: A system for applying energy to cells so as to elicit the formation of pores, to enhance transfection, and/or cell transformation, includes a computer, a plurality of acoustic probes for controllably applying acoustic energy to batches of cells, and a robot operatively for effecting relative movement between the probes and the batches of cells. Preferably, the acoustic energy comprises ultrasonic energy, which is applied in combination with optical or electrical energy to enhance the formation of pores in surface membranes of the cells.

Abstract: The invention concerns a method for treating an aqueous flow colonized by cells with a pulsed electric field applied to a flow, characterized in that the applied field is substantially parallel to the direction of flow and to its application to the transfer of nucleic acids (RNA, DNA, oligonucleotides) into cells, to the transfer of proteins to cells, to the extraction of cytoplasmic macromolecules and molecules contained in the cells, to cell fusion and the production of hybrids and/or to insertion of membrane proteins. It also concerns an electropulsing chamber, a method for destroying cells and a membrane permeabilization method.

Abstract: The present invention is related to methods in which an electric field pulse is applied to cells and tissue. Several embodiments of the present invention relate to the application of electric field pulses to cells to regulate the physiology and biophysical properties of various cell types, including terminally differentiated and rapidly dividing cells. Methods of regulating transcription of a gene in a cell, marking a cell for diagnostic or therapeutic procedures, determining cellular tolerance to electroperturbation, selectively electroperturbing a population of cells, reducing proliferation of rapidly dividing cells in a patient, and facilitating entry of a diagnostic or therapeutic agent into a cell's intracellular structures are also provided.

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: This invention provides improved electroporation methods for transferring nucleic acids of interest into host cells, wherein the host cells are (1) suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative, (2) mixed with the nucleic acids of interest, and (3) electrically treated. Also, this invention provides for kits used in the method for transferring nucleic acids into host cells.

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: A gene is transferred to the early stage chicken embryo by electroporation with placing the electrode tips to hold both ends of the embryo. Transfer of the desired gene to the early stage embryo was detected using the &bgr;-galactosidase gene of E coli as a reporter gene. The expression of the gene in the early stage embryo of chicken was thus confirmed. This method for transferring a gene into undifferentiated cells is simple to implement, highly efficient in transferring a gene, and applicable to various animal species.