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  • 1
    Electronic Resource
    Electronic Resource
    Transient transfection induces different intracellular calcium signaling in CHO K1 versus HEK 293 cells (2000)
    Springer
    Cytotechnology 33 (2000), S. 139-145 
    Details
    ISSN: 1573-0778
    Keywords: calcium phosphate ; CHO-K1 ; cytosolic calcium signaling ; HEK 293 ; laser scanning confocal microscopy ; transient transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract For the controlled production of recombinant proteinsin mammalian cells by transient transfection, it maybe desirable not only to manipulate, but also todiagnose the expression success early. Here, weapplied laser scanning confocal microscopy to monitortransfection induced intracellular Ca2+responses. We compared Chinese hamster ovary (CHO K1)versus human embryo kidney (HEK) 293 cell lines, whichdiffer largely in their transfectability. An improvedcalcium phosphate transfection method was used for itssimplicity and its demonstrated upscale potential.Cytosolic Ca2+ signaling appeared to inverselyreflect the cellular transfection fate. Virtually allCHO cells exhibited asynchronous, cytosolicCa2+ oscillations, which peaked 4 h afteraddition of the transfecting solution. Yet, most ofthe HEK cells displayed a slow and continuousCa2+ increase over the time of transfection. CHOcells, when exposed to a transfection-enhancingglycerol shock, strongly downregulated their Ca2+response, including its oscillations. When treatedwith thapsigargin, a Ca2+ store depleting drug,the number of successfully transfected CHO cells was significantly reduced. Our result points tointracellular store release as a critical componentfor the transfection fate of CHO cells, and its early detection before product visualization.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008150402616
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  • 2
    Electronic Resource
    Electronic Resource
    Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System (2000)
    Springer
    Cytotechnology 32 (2000), S. 199-208 
    Details
    ISSN: 1573-0778
    Keywords: 5-HT3 receptor ; large scale ; reactor ; Semliki Forest virus ; suspension process ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT3 receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A VibromixTM agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 106 cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT3 receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008192709549
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  • 3
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    Quantitative determination of large structures by small-angle light scattering (2001)
    Springer
    Details
    Publication Date: 2001-02-05
    Print ISSN: 0372-820X
    Electronic ISSN: 1435-1536
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Published by Springer
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