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Optimization of transduction of chosen cell lines using adenoviral vetors of the first generation

100%
Stopa M., Dulak A., Jozkowicz A.
|
2007
|
issue 3
123-140
EN
Efficacy of adenoviral vectors (AdV) was checked by transduction of six different cell lines (COS-7, HUVEC, HMEC-1, NIH 3T3, HaCaT, and B16(F10)) with the vectors containing reporter gene beta-galactosidase (Adbeta-gal). We optimised the adsorption time and dose of Ad beta-gal. The transduction with efficacy of up to 100% was obtained for the doses 10-100 IU/cell. In order to examine the effect of transduction procedure on biology of the cells, we measured the production of major proinflammatory cytokines. In several cell lines (HMEC-1, HUVEC, HaCaT), we found the induction of IL-6 synthesis and decrease in the cell viability, particularly in the case of the highest Adbeta-gal dose. However, the treatment with adenoviral vectors did not induce TNF generation and did not modify proliferation of cells.
2

The most important features of adenoviral vectors

100%
Stopa M., Dulak J., Jozkowicz A.
|
2007
|
issue 3
22-32
EN
One of the major hurdles to successful gene therapy is the ability to efficiently introduce a foreign nucleic acid into the tissue of interest. As adenoviruses posses ability to enter rapidly a mammalian host cell and achieve propagation, they are widely used as a tool for delivery transgenes. Recombinant adenoviral vectors of first generation (AdV) contain an expression cassette with exogenous genes and are made replication deficient by the deletion of the E1/E3 region. They offer many advantages for gene delivery: ability to transduce a wide variety of cell types in a cell-cycle independent manner, easy and cheap propagation process to high titers and low pathogenicity for humans. However, AdV also have some disadvantages, namely cytotoxity, immunogenity, transient expression of transgene, which are mostly important in case of clinical trials. Despite those limitations and development of more sophisticated adenoviral systems (helper-dependent adenoviral vectors), AdV of first generation are still widely used for trasnducting different cell types in vitro, especially those that are refractory to other gene transfer methods, as well as in gene therapy clinical trials.
3

Production of adenoviral vectors of first generation ? optimization of method

88%
Stopa M., Jozwa A., Mleczko J., Dulak A., Jozkowicz A.
|
2007
|
issue 3
98-122
EN
The paper presents the production of adenoviral vectors (AdV) containing beta-galactosidase (Adbeta-gal), from the transfer of recombinant viral DNA into packing cell line (HEK293) to the titration of viral particles. Optimisation of the methods (preparation of DNA for transfection, adsorption time during the infection of cells, amount of serum in the medium, time-point of vector isolation) enables obtaining a titer of up to 1010 IU/mL. Adbeta-gal were titrated with several methods, with beta-gal in situ staining used as a reference. We found that the most suitable titration method of the vectors containing other than reporter genes was the Rapid Titer ELISA kit?.
4
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Denoising methods for improving automatic segmentation in OCT images of human eye

76%
Stankiewicz A., Marciniak T., Dąbrowski A., Stopa M., Rakowicz P., Marciniak E.
|
2017
|
issue 1
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