Abstract: The present invention describes new mammalian expression vectors comprising a novel combination of regulatory elements and one or more selection marker gene(s). The vector allows for incorporation of at least one, preferably two or more genes of interest, its/their subsequent expression, and for selection of transfected cells using, e.g., G418 and/or MTX. The pDGP?GOI vector as an example for a mammalian expression vector according to the present invention exhibits a 9555 bp sequence, one strand of which is represented by SEQ ID NO:2.

Abstract: The present invention relates to mutated tissue plasminogen activators, and their use for treating thrombotic diseases.

Abstract: A variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue corresponding to any of amino acids 4, 6, 13, 14, 15, 16, 20, 45, 47, 51, 54, 61, 68, 69, 70, 71, 72, 73, 74, 75, 77, 78, 80, 82, 87, 88, 94, 95, 100, 103, 104, 117, 124, 125, 126, 128, 130, 133, 134, 136, 143, 144, 146, 168, 174, 177, 178, 183, 186, 188, 189, 190, 194, 195, 199, 200, 201, 204, 207, 210, 214, 217, 219, 222, 225, 227, 233, 234, 235, 236, 240, 251, 252, 254, 258, 259, 260, 261, 262, 263, 264, 266, 267, 269, 271, 273, 281, 282, 283, 284, 286, 288, 299, 322, 323, 325, 327, 328, 331, 334, 350, 356, 358, 367, 370, 371, 374, 377, 378, 388, 391, 414, 421, 422, 445, 450, 467, 488, 505, 536, 548, 583, 588, 603, 637, 648, 651, 652, 660, 676, 677, said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered pr

Abstract: The present invention relates to mutated tissue plasminogen activators, and their use for treating thrombotic diseases.

Abstract: The present invention relates to a host cell comprising a cofilin-specific small interfering RNA (siRNA) sequence. The host cell may further comprise a nucleic acid encoding a recombinant protein. The present invention also relates to a method for producing a recombinant protein by the host cell comprising a cofilin-specific small interfering RNA (siRNA) sequence.

Abstract: The present invention relates to a host cell comprising a cofilin-specific small interfering RNA (siRNA) sequence. The host cell may further comprise a nucleic acid encoding a recombinant protein. The present invention also relates to a method for producing a recombinant protein by the host cell comprising a cofilin-specific small interfering RNA (siRNA) sequence.

Abstract: The present invention relates to a method for identification of anti-fungal agents and their mode of actions. In particular, it relates to cell wall disturbing anti-fungal agents and to an assay to identify them. More particularly, it relates to a screening method for the identification of an anti-fungal compound, which method comprises (i) contacting a potential anti-fungal compound with a polypeptide which is involved in cell wall synthesis; and then (ii) identifying the effect which the potential anti-fungal compound has on the activity of the polypeptide, whereby reduced polypeptide activity is indicative for anti-fungal activity of the potential anti-fungal compound. It also relates to an overexpressing host cell and to a kit for performing the assay.

Abstract: The invention relates to isolation of novel ?-actin and ribosomal protein S21 (rpS21) promoters and uses thereof. In particular, this invention features nucleotide sequences for rodent ?-actin promoters including, hamster, rat, and mouse, and hamster rpS21 promoter.

Abstract: In order to make observation of localizations of in vivo expressed genes possible in an entire individual living organism such as animals, and plants, a living genetic recombinant specimen containing a marker that can be detected at the time when specific genes are expressed is sequentially severed, sectional images each of which corresponds to an image of sections severed are photographed in every scissions of the living specimen, and three-dimensional observation of the living specimen is implemented on the basis of the images photographed in every scissions described above, whereby three-dimensional localizations of expressed genes are observed in the living specimen.

Abstract: The invention provides improved methods of recombinant protein production in cell culture. More specifically, the invention relates to the modulation of the IGF-1 signaling pathway in cells so as to improve production characteristics.

Abstract: The invention provides cells that produce increased levels of recombinant protein by modulating the activity of translational regulator gene products that are downstream targets of PKB alpha, methods of making such cells, and methods of using such cells. Such translational regulator gene products include 4E-BP1 and mTOR.

Abstract: A cell and tissue culture modeling device comprising a housing having an interior chamber, an inlet port in fluid communication with the internal chamber, an outlet port in fluid communication with the internal chamber, a plurality of hollow fibers disposed within the interior chamber and traversing the length of the housing between the inlet port and the outlet port. Each of the plurality of hollow fibers has an interior defining an intracapillary space and the interior chamber defines an extracapillary space unoccupied by the plurality of hollow fibers. To access the extracapillary space of the device, a portion of the housing is removable. The device can be used to conduct permeability, drug efficacy, and gene expression studies.

Abstract: A glycoprotein is produced by a process comprising culturing mammalian host cells expressing nucleic acid encoding said glycoprotein in the presence of (a) a factor that modifies growth state in a cell culture, (b) a divalent metal cation that can adopt and prefers an octahedral coordination geometry, and/or (c) a plasma component. In this process, the occupancy of an N-linked glycosylation site occupied only in a fraction of a glycoprotein is enhanced. Such culturing is preferably carried out at a temperature of between about 30° C. and 35° C. and/or in the presence of up to about 2 mM of a butyrate salt and/or in the presence of a cell-cycle inhibitor.

Abstract: The invention provides improved methods of recombinant protein production in cell culture. More specifically, the invention relates to the modulation of the IGF-1 signaling pathway in cells so as to improve production characteristics.

Abstract: A recombinant DNA molecule adapted for transfection of a host cell comprising a nucleic acid molecule encoding mammalian erythropoietin or tissue plasminogen activator, an expression control sequence operatively linked thereto and at least one SAR element. The invention also relates to expression vectors having the recombinant DNA molecule and to mammalian cells transformed with the expression vector. The mammalian cells lack multiple copies of an amplified amplification gene and are capable of expressing recombinant EPO or tPA in vitro at levels of at least 1,500 u or 500 u/10.sup.6 cells in 24 hours respectively. The invention further relates to a method of expressing recombinant mammalian EPO or tPA using the expression vectors and to a transgenic non-human animal or embryo whose germ cells and somatic cells contain a DNA construct having the recombinant DNA molecule of the invention.

Abstract: A method for producing tissue plasminogen activator (t-PA) in eukaryotic host cells is disclosed. Enhanced levels of t-PA production are obtained by co-amplification of the t-PA gene through treatment of cultures transformed with mutant or wild type DHFR with methotrexate.

Abstract: Tissue plasminogen activator (t-PA) derivatives are produced in useful quantities using recombinant DNA techniques. Specific derivatives include amino acid deletion derivatives and amino acid substitution derivatives. A deletion derivative lacking the N-terminal first 68 amino acids is specifically exemplified having requisite t-PA characteristics. The invention disclosed thus enables the production of t-PA derivatives via recombinant means. Methods, expression vehicles and various host cells useful in the production of said t-PA derivatives are also disclosed.

Abstract: Tissue plasminogen activator (t-PA) derivatives are produced in useful quantities using recombinant DNA techniques. Specific derivatives include amino acid deletion derivatives and amino acid substitution derivatives. A deletion derivative lacking the N-terminal first 68 amino acids is specifically exemplified having requisite t-PA characteristics. The invention disclosed thus enables the production of t-PA derivatives via recombinant means. Methods, expression vehicles and various host cells useful in the production of said t-PA derivatives are also disclosed.

Abstract: The invention provides expression vectors containing the promoter, enhancer and substantially complete 5'-untranslated region including the first intron of the major immediate early gene of human cytomegalovirus. Further vectors including the hCMV-MIE DNA linked directly to the coding sequence of a heterologous gene are described. Host cells transfected with the vectors and a process for producing heterologous polypeptides using the vectors and the use of the hCMV-MIE DNA for expression of a heterologous gene are also included within the invention.

Abstract: The present invention provides derivatives of tissue plasminogen activator that lack the Finger, Growth Factor and Kringle 1 domains and comprise a Kringle 2 domain that is monoglycosylated at a site other than that of t-PA. Using recombinant DNA techniques, an alternate glycosylation sequence is provided within the Kringle 2 domain of these t-PA derivatives. This alternate glycosylation consensus sequence, as well as the glycosylation consensus sequence within the Serine Protease domain, is glycosylated upon the expression and secretion of these molecules from eucaryotic host cells. Thus, a homogeneous population of diglycosylated t-PA derivatives that lack the Finger, Growth Factor and Kringle 1 domains is produced.

Abstract: A hybrid vector carrying a first and second DNA segments operationally linked thereto, the first DNA segment encoding a protein capable of cross-linking to the cap structure of mRNA and mediating ribosome-binding, and the second DNA segment encoding a polypeptide or protein, the vector being capable of replication, transcription and translation to express the factor and the polypeptide or protein upon transformation of a eukaryotic host, and the polypeptide or protein being expressed at a level higher than the level of expression thereof in the absence of the first DNA segment. A eukaryotic host is transformed with this hybrid vector. Also disclosed is a method of increasing the synthesis of a polypeptide or protein in a eukaryotic host cell.

Abstract: DNA constructs useful in the production of thrombopoietin are disclosed. In general, the DNA constructs comprise a first DNA segment encoding a fusion of an amino-terminal secretory peptide joined to a thrombopoietin polypeptide and one or more additional DNA segments that provide for the transcription of the first segment. The secretory peptide is a native mammalian t-PA secretory peptide or may be modified to enhance proteolytic cleavage of the fusion. Also disclosed are cultured eukaryotic cells containing these DNA constructs and methods for producing thrombopoietin polypeptides through the use of the DNA constructs and cultured eukaryotic cells.

Abstract: Tissue plasminogen activator (t-PA) zymogens and variants are prepared, including a fibrinolytically active variant of t-PA that has an amino acid alteration at a site within the protease domain of t-PA as compared with the corresponding wild-type t-PA, which alteration renders the variant zymogenic in the presence of plasmin-degraded fibrinogen, and/or fibrin (or plasma clot) specific, as compared to the corresponding wild-type t-PA. DNA sequences can be prepared that encode the zymogens and variants, as well as expression vectors incorporating the DNA sequences, and host cells transformed with the expression vectors. The zymogens and variants may be used in a pharmaceutical preparation to treat a vascular disease or condition or to prevent fibrin deposition or adhesion formation or reformation in mammals.

Abstract: Tissue plasminogen activator (t-PA) zymogens and variants are prepared, including a fibrinolytically active variant of t-PA that has an amino acid alteration at a site within the protease domain of t-PA as compared with the corresponding wild-type t-PA, which alteration renders the variant zymogenic in the presence of plasmin-degraded fibrinogen, and/or fibrin (or plasma clot) specific, as compared to the corresponding wild-type t-PA. DNA sequences can be prepared that encode the zymogens and variants, as well as expression vectors incorporating the DNA sequences, and host cells transformed with the expression vectors. The zymogens and variants may be used in a pharmaceutical preparation to treat a vascular disease or condition or to prevent fibrin deposition or adhesion formation or reformation in mammals.

Abstract: A biochemically defined culture medium for culturing engineered Chinese hamster ovary (CHO) cell lines, which is essentially free from protein, lipid and carbohydrate isolated from an animal source, having water, an osmolality regulator, a buffer, an energy source, amino acids including L-glutamine, an inorganic or recombinant iron source, and a synthetic or recombinant growth factor, and optionally non-ferrous metal ions vitamins and cofactors. Also cells adapted to grow in such a culture medium. REEXAMINATION RESULTS The questions raised in reexamination request no. 90/006656, filed Jun. 2, 2003 have been considered and the results thereof are reflected in this reissue patent which constitutes the reexamination certificate required by 35 U.S.C. 307 as provided in 37 CFR 1.570(e), for ex parte reexaminations, or the reexamination certificate required by 35 U.S.C. 316 as provided in 37 CFR 1.997(e) for inter partes reexaminations.