Abstract: The present invention relates to protein expression systems and in particular to a mammalian protein expression system. Specifically, the present invention provides a rodent cell line with enhanced protein production capabilities. The invention also relates to eukaryotic cloning and expression vectors and related methods, and in particular to DNA vectors capable of high level expression of a protein of interest. The invention allows for long-term episomal maintenance of expression vectors in mammalian cells.

Abstract: One or more cells can be cultured when confined in space by barriers. The distance between barriers can be comparable to the size of a cell to be cultured. The space between barriers can also be sufficiently small to allow control of cell properties or monitoring of the cell(s) cultured therein. The cell(s) may be confined completely or may be mobile between two opposing barrier surfaces. The gap between two opposing barriers may be sufficiently narrow to allow only a monolayer of cells to be cultured. A barrier can be transparent. The surfaces of the barriers may have one or more pre-selected characteristics that mimic the characteristics of a biological niche of the cells(s). The number of cells in a cell culture may be limited to permit control of properties of individual cells. The cultured cell(s) may be monitored, such as imaged, over a long period of time, using standard bright field or fluorescent imaging techniques.

Abstract: The present invention provides a process for isolating vascular endothelial cells from embryoid bodies differentiated from embryonic stem cells, which comprises: (a) treating embryoid bodies differentiated from embryonic stem cells with 0.005-0.015% trypsin and 0.05-0.15 mM ethylenediaminetetraacetate (EDTA) to obtain vascular endothelial cell clusters; and (b) treating the vascular endothelial cell clusters with 0.1-0.5% trypsin and 0.5-2 mM EDTA so as to separate the vascular endothelial cell clusters into single cells.

Abstract: The present invention relates to an electrofusion microelectrode made of a tube having a first proximal end and a second distal end. The tube has an electrically conductive coating on its exterior surface that extends continually from the first proximal end of the tube toward the second distal end of the tube. Also disclosed is an electrofusion microelectrode unit having an electrofusion microelectrode and a holding tool capable of receiving the electrofusion microelectrode at the second distal end of the tube. The present invention also relates to a system having two or more electrofusion microelectrodes of the present invention and to methods of manipulating cells and/or cellular components using the electrofusion microelectrodes, units, and systems of the present invention.

Abstract: The present invention provides adapter-directed display systems for expressing exogenous polypeptide within a host cell and/or displaying the exogenous polypeptide on the outer surface of a genetic package. This subject systems are particularly useful for displaying a genetically diverse repertoire of monomeric and multimeric polypeptides. The invention also provides both expression and helper vectors and kits containing components of the subject display systems. Also provided are genetic packages displaying the exogenous polypeptides of particular interest. Further provided by the invention are methods of using the subject display systems.

Abstract: Novel isolated polynucleotides and polypeptides associated with the lignin biosynthetic pathway are provided, together with genetic constructs including such sequences. Methods for the modulation of lignin content, lignin structure and lignin composition in target organisms are also disclosed, the methods comprising incorporating one or more of the polynucleotides of the present invention into the genome of a target organism.

Abstract: Compositions and methods are provided for screening for compounds that modulate insulin promoter activity. Vectors that express green fluorescent protein under the control of the human insulin promoter are introduced into mouse and human cells in which the insulin promoter is expressed in a glucose-responsive manner. Such cells are then used to screen for compounds that modulate insulin promoter activity.

Abstract: The present invention relates to the field glycosylation engineering of proteins. More particular, the present invention is directed to the glycosylation engineering of proteins to provide proteins with improved therapeutic properties, e.g., antibodies, antibody fragments, or a fusion protein that includes a region equivalent to the Fc region of an immunoglobulin, with enhanced Fc-mediated cellular cytotoxicity.

Abstract: The present invention discloses a method for modulating the production of a protein from a polynucleotide in a CHO cell by replacing at least one codon of the polynucleotide with a synonymous codon that has a higher or lower translation efficiency in the CHO cell than the codon it replaces, or by introducing into the CHO cell a polynucleotide that codes for an iso-tRNA which limits the rate of production of the polypeptide and which corresponds to a codon of the first polynucleotide. The present invention also discloses the use of a protein-encoding polynucleotide whose codon composition has been modified for enhanced production of the protein in CHO cells.

Abstract: Described is the production of proteins in a host cell. More specifically, described are methods for improving expression of two or more proteins in a cell or host cell. The methods are suited for production of, for example, recombinant antibodies that can be used in pharmaceutical preparations or as diagnostic tools. In one embodiment, provided is a method for obtaining a cell that expresses two or more proteins comprising providing the cell with two or more protein expression units encoding two or more proteins, characterized in that at least two of the protein expression units comprise at least one STAR sequence.

Abstract: Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.

Abstract: This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes.

Abstract: The present invention provides a novel apparatus to grow cells where the cultivation chamber (1) is partially filled with liquid cultivation medium and cells. Mixing and aeration is achieved by generating intermittently one single large gas bubble (6) at the bottom of the column bioreactor, the single large bubble width representing from 50 to 99% of the tank width, preferably from 60 to 99%, more preferably 98.5%. The culture medium flows out as a film between the large bubble and the inner wall of the bioreactor. This rising bubble allows mixing and aeration of the bulk. As the design of the invention is very simple, it is possible to manufacture it with flexible plastic material and use the apparatus as a disposable system. Moreover, such a mixing/aeration principle minimizes cell damages usually due to shear stress and small bubbles and allows easy and efficient scale-up from small scale to a larger one. Such a large-scale, efficient and disposable culture system can largely reduce production costs.

Abstract: The invention features methods of analyzing the kinetics properties of transfection reactions. Also featured are methods for creating structural promoters which are effectively unregulated by enhancers and repressors. The structural promoters are significantly more active than the native promoter sequences upon which they are based.

Abstract: A microinjection apparatus has a trap plate which traps at least one cell, so as to fix a position thereof, a capillary needle which injects a substance into the cell trapped by the trap plate, a movement mechanism portion which thrusts a distal end of the capillary needle onto a in a lengthwise direction of the needle, and which withdraws the capillary needle in the lengthwise direction thereof, and a discharge control portion which discharges the substance from the distal end of the capillary needle, when the capillary needle has been withdrawn to a predetermined position by the movement mechanism portion.

Abstract: The present invention relates to the simple, gentle, and efficient extraction of biological material from Escherichia coli (E. coli). The use of E. coli in research laboratories depends on the ability to prepare lysates to isolate the desired products under investigation. The present invention includes methods and engineered E. coli strains that are capable of rapid controlled lysis or herein “autolysis”. The XJa strains were made from JM109 and the XJb strains from BL21 by insertion of the ? R or (? SR) lytic endolysin gene to replace the tightly regulated araB gene. Thus, arabinose becomes a non-metabolizable inducer and the controlled autolysis phenotype is induced by the PBAD promoter by the presence of saturating arabinose. Upon induction of the bacteriophage ?R endolysin, the E. coli remains intact but is efficiently lysed after one freeze-thaw cycle. The present invention is usable with many different buffer systems and is flexible in this regard.

Abstract: The invention relates to an optical method for targeted transfer of molecules, preferably of DNA, RNA, peptides, amino acids and proteins, into vital cells by means of laser radiation and to an arrangement for implementing the method. The object of the invention, to find a novel possibility for targeted molecule transfer into the interior of vital cells, particularly the transfer of DNA, RNA, peptides, amino acids and proteins, which achieves a high transfer efficiency while extensively excluding destructive side effects such as a lethal effect on a treated cell, is met according to the invention in that cellular membranes are opened transiently for the molecule transfer by multiple laser pulses in the microjoule range or less and a pulsed, near-infrared laser beam with a pulse width in the femtosecond range is directed in each instance to a submicrometer spot of a membrane of the vital cell for an irradiation period of less than one second.

Abstract: The present invention is based, in part, on the discovery that parvovirus (including AAV) capsids can be engineered to incorporate small, selective regions from other parvoviruses that confer desirable properties. The inventors have discovered that in some cases as little as a single amino acid insertion or substitution from a first parvovirus (e.g., an AAV) into the capsid structure of another parvovirus (e.g., an AAV) to create a chimeric parvovirus is sufficient to confer one or more of the desirable properties of the first parvovirus to the resulting chimeric parvovirus and/or to confer a property that is not exhibited by the first parvovirus or is present to a lesser extent.

Abstract: The capillary gun for delivery of ballistic particles to a target includes an inner capillary tube disposed concentrically within an outer capillary tube with the input end of the inner tube connected to a channel through which a continuous flow of high speed helium gas carrying ballistic particles is introduced. The outer capillary tube, which is connected to a vacuum source, has an outlet end that extends slightly beyond the end of the inner tube. A cap placed over the output end of the outer tube has an opening at its center through which the particles exit the device. The vacuum source applies continuous suction to the space between the outer tube and the inner tube, drawing the gas from the output end of the inner tube while the inertia of the accelerated particles causes them to continue in the axial direction through the exit opening for delivery to the target. Multiple particle injectors provide for the concurrent injection of different materials without disruption of the gas flow.

Abstract: This invention provides novel enzyme compositions using newly identified and isolated C. lucknowense enzymes, including CBH Ib CBH IIb, EG II, EG VI, ?-glucosidase, and xylanase II in conjunction with previously identified enzymes CBH Ia, CBH IIa (previously described as Endo 43), and EG V. These enzyme compositions demonstrate an extremely high ability to convert lignocellulosic biomass (e.g., Avicel, cotton, Douglas fir wood pretreated by organosolv) to glucose. CBH Ia and IIb, which both have a cellulose-binding module (CBM) displayed a pronounced synergism with three major endoglucanases (EG II, EG V, EG VI) from the same fungus in hydrolysis of cotton as well as a strong synergy with each other. The enzyme compositions are effective in hydrolysis of the lignocellulosic biomass.