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 invention provides a method of transferring in vivo a molecule into a striated muscle cell. More specifically, a method of the invention comprises contacting in vivo a striated muscle cell with a molecule, and electrically stimulating the muscle cell with one or more unipolar pulses of an electric field intensity ranging from 1 to 800 V/cm2. In one embodiment, the molecule is a nucleic acid encoding a protein of interest. For example, the invention provides methods of promoting angiogenesis and hemostasis.

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: 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: The present invention provides a method for enhancing the in vivo delivery of chimeric oligonucleotides, containing for example DNA/2′OMeRNA, into cells of a plant, an animal or a human, comprising a step of applying topically to or injecting into a tissue, or tissue adjacent to a tissue, containing said cells, a composition comprising said chimeric oligonucleotide, followed by, preceded by, or simultaneously to a step of transferring said chimeric oligonucleotide into said cells by iontophoresis, and relates to a gene therapy method comprising the iontophorically transfer of a chimeric oligonucleotide DNA/2′OMeRNA. The present invention is also directed to particular chimeric oligonucleotides DNA/2′OMeRNA capable of inducing or inhibiting the expression of a specific gene involved in eye function by inducing or reverting a mutation in that specific gene, and their use as therapeutic composition for preventing or treating ocular diseases.

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: The invention concerns an improved method for transferring in vivo multicelled eukaryotic organism cells nucleic acids or nucleic acids combined with products for enhancing the efficacy of such transfers. The invention also concerns the combination of a nucleic acid and the transfer method for use in gene therapy.

Abstract: A process for providing a recombinant, heterologous gene in the genome of a eukaryotic cell is provided. In particular, heterologous DNA is inserted into a recipient gene of the eukaryotic cell by homologous recombination. Moreover, a transgenic or chimeric animal comprising cells with DNA inserted into its genome by homologous recombination is disclosed. Further, the method of making these transgenic animal is taught.

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 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: An apparatus and method for controlled release of nucleotides from electrodes utilizing potential difference for use in delivering the nucleotide into an organism. The frequency, duration and rate of release may be specifically controlled to obtain optimal release of the nucleotide. The apparatus and method may be used in conjunction with methods for electroporation, wherein the nucleotide may transit the cell membrane, and move into the cell. Alternatively, the apparatus and method may be used in conjunction with complexes of DNA and lipids or other carriers, which complexes will transit the cell membrane and move into the cell. The apparatus and method may be used for gene therapy, for use in treatment of any of a wide variety of diseases.

Abstract: The invention comprises an apparatus and a method of effecting localized electroporation in a relatively small target area and for introducing foreign matter into cellular material in which the target area is located. The cellular material may be in vitro, in ovo or in vivo.

Abstract: The present invention provides a chimeric protein which comprises a mutated DNA methyltransferase portion and a DNA binding protein portion that binds sufficiently close to a promoter sequence of a target gene which promoter sequence contains a methylation site, to specifically methylate the site and inhibit activity of the promoter and thus inhibit expression of the target gene. This invention also provides for a method for inhibiting the expression of a target gene which includes contacting a promoter of the target gene with the chimeric protein, so as to specifically methylate the promoter sequence of the target gene thus inhibiting expression of the target gene.

Abstract: An electroporation and/or fusion treatment of microscopic objects occurs in a medium between at least two electrodes, with the electrodes being miniaturized electrodes in a microsystem with a channel structure which is set up for the flow-through of the medium with the objects.

Abstract: The present invention involves the role of p53 in the differentiation of embryonic tissues. More particularly, the present invention provides methods of the blocking of p53 function in embryonic tissues, and the use of these tissues as screening tools for substances that are capable of overcoming the p53-related block in differentation, both in vitro and in vivo. The similarities between undifferentiated embryonic cells and tumor cells is evident, and thus these assays serve as a model for possible cancer therapeutics. In addition, methods for identifying additional cellular components that interact p53 or p53-related pathways are provided.

Abstract: The present invention relates to a human or mammalian DNA replication origin consensus sequence which consists of a sequence selected from the group consisting of CCTMDAWKSGBYTSMAAWYWBCMYTTRSCAAATTCC (SEQ ID NO: 1); and AWMTWAAKRAWRWWKKDAVWWGAKRWWKWVWHRASSACMDWKAAKTWKGGWTWARRYWKGRKMWWTWKAWSDATAKWWWKDAKWKMWRKTT (SEQ ID NO: 4). A method for the control of initiation of mammalian DNA replication which comprises the steps of: a) inserting a consensus sequence coding for a sequence of the present invention together with a DNA fragment to form a vector capable of expression of the DNA fragment; b) introducing the vector of step a) into mammalian cells in vitro.

Abstract: An electroporation apparatus for introducing exogenous material into cells is described herein. The apparatus comprises first a base member configured for holding a cell support, the cell support having a top surface portion, with the top surface portion configured for carrying adherent cells. The apparatus further comprises an electrode carrier operably associated with the base member, the electrode carrier having a bottom surface portion, a first electrode connected to the electrode carrier, and a second electrode also connected to the electrode carrier. The electrode carrier has a channel formed therein, with the channel positioned between the first electrode and the second electrode, so that exogenous material may be introduced through the channel and into contact with the cells. Methods for introducing exogenous compounds into a cell and for visually detecting the location of binding events within a cell are also disclosed.

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: 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: A method of expression DNA in a cell comprises, in a cell that expresses a replication VpA factor, transfecting the cell with a vector, wherein (i) the vector contains a DNA, or is adapted to receive a DNA, in operative combination with a promoter for expression of the DNA; and (ii) extrachromosomal replication of the vector is dependent upon presence within the cell of the replication factor. Stable El maintenance of the vector and expression of the DNA is thereby achieved. Vectors for expression of DNA are provided as are assays for the effect of expression of DNAs in cells, the DNAs being H expressed from the vectors.

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: The invention relates to methods and reagents for influencing alternative RNA splicing in living cells. More particularly, the invention relates to novel means for influencing RNA splice choice in living cells using polynucleotide-based reagents that compete for binding sites in nucleotide binding proteins, and novel methods for using these reagents as therapeutics.

Abstract: This invention relates to a novel method of in vivo methylation.

Abstract: Disclosed are methods for the isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, methods for transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.

Abstract: A new and useful apparatus for producing cell electrofusion is provided. The apparatus comprises: a. a chamber with a substrate disposed therein, b. means for directing the cells to be fused toward one side of the substrate; and c. a device for inducing fusion of the portion of the cells.

Abstract: The present invention provides methods and apparatus for the delivery of at least one chromosome into a cell. Invention methods and apparatus employ FACS or MACS technology for rapidly processing cells and for confirming the introduction of chromosome(s) into a cell. The introduction of the chromosome(s) into the cell is mediated by one or more of a laser, a linear accelerator or electrically induced fusion of a cell and encapsulated chromosome(s). Invention methods provide for the rapid and reliable processing associated with FACS and MACS technology to process thousands of cells a minute, thereby enabling large scale gene transfer.

Abstract: The invention concerns an improved method for transferring in vivo multicelled eukaryotic organism cells nucleic acids or nucleic acids combined with products for enhancing the efficacy of such transfers. The invention also concerns the combination of a nucleic acid and the transfer method for use in gene therapy.

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: 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: The present invention relates to a method for introducing nucleic acids into cells for e.g. producing transiently transfected or stably transformed animal cells by using a specifically designed nucleic acid/protein complex comprising in operable linkage to an expressible DNA or to an oligonucleotide a VirD2 protein, preferably together with a VirE2 protein.

Abstract: A loading system for providing a cell suitable for delivery of an agent to a vertebrate, the system comprise a loading module for loading a cell with an agent; and a sensitization module in fluid communication with the loading module, the sensitization module for sensitizing a cell to an energy field, such that said cell is induced to release the agent upon exposure to said energy field. The system can be used to transform a cell, such as a red blood cell, into a delivery vehicle for delivering a therapeutic agent and/or an imaging agent to a vertebrate, and in particular, to a mammal, such as a human being.

Abstract: The present invention relates to transfected primary and secondary somatic cells of vertebrate origin, particularly mammalian origin, transfected with exogenous genetic material (DNA) which encodes a desired (e.g., a therapeutic) product or is itself a desired (e.g., therapeutic) product, methods by which primary and secondary cells are transfected to include exogenous genetic material, methods of producing clonal cell strains or heterogenous cell strains, 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: A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a transposase protein and (b) a polynucleotide that comprises (1) a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and (2) a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

Abstract: Disclosed are methods for the isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, methods for transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.

Abstract: A modified Factor VIII cDNA is described comprising deletion of the B-domain and insertion of at least one truncated Factor IX intron 1 in the Factor VIII cDNA. The modified Factor VIII cDNA may be used for the production of a high yield of Factor VIII in vitro. The modified Factor VIII cDNA may be incorporated into a vector for gene therapy.

Abstract: An electroporation system and method for directing high-voltage currents to a suspension of cells contained in a cuvette. A high-voltage switch controls the coupling of a charge control to a high-voltage capacitor and, additionally, controls the coupling of the high-voltage capacitor to the cuvette. A current diverter diverts current away from the sample whenever an arc condition commences or a low sample resistance is detected across the cuvette. The current diverter includes a current diverter switch, which is triggered when a sense resistor measures a second predetermined voltage indicative of an arc-over event (or a low sample resistance condition).

Abstract: The invention relates to methods for targeting an exogenous polynucleotide or exogenous complementary polynucleotide pair to a predetermined endogenous DNA target sequence in a eukaryotic cell by homologous pairing, particularly for altering an endogenous DNA sequence, such as a chromosomal DNA sequence, typically by targeted homologous recombination. In certain embodiments, the invention relates to methods for targeting an exogenous polynucleotide having a linked chemical substituent to a predetermined endogenous DNA sequence in a metabolically active eukaryotic cell, generating a DNA sequence-specific targeting of one or more chemical substituents in an intact nucleus of a metabolically active eukaryotic cell, generally for purposes of altering a predetermined endogenous DNA sequence in the cell.

Abstract: A transgenic mouse with alterations in an abc1 gene is prepared by introduction of an altered abc1 gene into a host animal. The resulting transgenic mice do not produce functional ABC1 protein molecules. Cells and cell lines derived from these animals also contain the altered abc1 gene.

Abstract: A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

Abstract: The invention relates to a process for preparing clonogenic fibroblasts, with tissue being removed from the donor and the individual cells being isolated from the tissue, the resulting cell suspension being strained, the cells which are contained in the cell suspension being washed and the cells being converted into a tissue culture, with the exception of the isolation of individual cells by mechanical comminution, followed by an enzymic treatment with collagenase alone, and with at least one gene being inserted into the fibroblasts by means of the transfection, which gene encodes a biologically active protein, preferably a therapeutically active protein, for example a growth factor, a hormone, an enzyme, a coagulation factor or a coagulation inhibitor.

Abstract: Transgenic mice with a non-functional proteinase activated receptor-2 (PAR2) gene are prepared by targeted disruption of the endogenous PAR2 gene. The resulting transgenic mice display a phenotype including a lack of a hypotensive response to administration of the peptide, SLIGRL, and a reduction in carrageenin-induced paw edema compared to wild type mice.

Abstract: Disclosed is substantially pure DNA encoding a C. elegans Egl-10 polypeptide; substantially pure Egl-10 polypeptide; methods of obtaining RGS encoding DNA and RGS polypeptides; and methods of using the RGS DNA and RGS polypeptides to regulate G-protein signalling.

Abstract: The present invention is a method for creating a localized disruption within a boundary of a cell or structure by exposing a boundary of a cell or structure to a set of energetically charged particles while regulating the energy of the charged particles so that the charged particles have an amount of kinetic energy sufficient to create a localized disruption within an area of the boundary of the cell or structure, then upon creation of the localized disruption, the amount of kinetic energy decreases to an amount insufficient to create further damage within the cell or structure beyond the boundary. The present invention is also a method for facilitating the introduction of a material into a cell or structure using the same methodology then further exciting the area of the boundary of the cell or structure where the localized disruption was created so to create a localized temporary opening within the boundary then further introducing the material through the temporary opening into the cell or structure.

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 transfected primary and secondary somatic cells of vertebrate origin, particularly mammalian origin, transfected with exogenous genetic material (DNA) which encodes a desired (e.g., a therapeutic) product or is itself a desired (e.g., therapeutic) product, methods by which primary and secondary cells are transfected to include exogenous genetic material, methods of producing clonal cell strains or heterogenous cell strains, 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: 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 computer system for controlling the parameters used during molecule transfer operations. The computer system includes a processor coupled to a memory. The memory stores instructions that are executed by the processor. The computer system controls the value of parameters that affect the characteristics of pulses delivered by a pulse generating circuit to a solution. The pulse generating unit delivers individual pulses during cycles. The cumulative pulse delivered during a specified number of cycles is a pulse output. The magnitude of a pulse output is determined by the number of cycles corresponding to the pulse output and the characteristics of the individual pulses delivered during the cycles. A pulse group is a series of pulse outputs. The computer system controls a molecule transfer operation by causing the pulse generation circuit to deliver one or more pulse groups to the solution. A user selects the shape and number of pulse groups to be delivered for an operation.