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Transient expression assay for optimization of direct gene transfer into cucumber meristem protoplasts by electroporation

100%
Burza W., Wochniak P., Wroblewski T., Malepszy S.
Journal of Applied Genetics
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1995
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tom 36
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nr 1
The paper presents a new way of obtaining viable and very homogeneous cucumber protoplasts. Protoplasts from cells formed in the shoot tip meristem culture were isolated from suspension. Plasmid pBI121 was introduced using impulse electric field. Effectiveness of transformation process was determined by the transient expression of ß-glucuronidase (GUS) gene, controlled by promotor 35S. The activity of ß-glucuronidase enzyme as a product of GUS reporter gene was estimated by fluorimetric method (JEFFERSON 1987). Parameters of electroporation process were optimized. The transient expression of GUS gene was measured 24 h after electroporation. The highest effectiveness of transformation process was achieved using three electric impulses at the initial voltage of 250-350 V at 10-sec. intervals as a result of discharging a 140 µF capacitor and 50-70 µg × cm⁻³ plasmid DNA in the presence of 50 µg × cm⁻³ carrier DNA. The system presented is an effective method of exogenic DNA transfer, which is indicated by a high transient expression of the reporter gene. In comparison to Agrobacterium tumefaciens and A. rhizogenes, this alternative method of gene transfer can be used for obtaining transgenic cucumber plants.
2

Effect of DNA methylation on transient expression of PR-s genes in tobacco protoplasts

100%
Brodzik R., Hennig J.
Journal of Applied Genetics
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1996
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tom 37A
3

Stable genetic transformation of mature wheat embryos using silicone carbide fibers and DNA imbibition

84%
Sawahel W., Saker M.
Cellular and Molecular Biology Letters
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1997
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tom 02
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nr 4
Improvement of wheat (Triticum aestivum L) by biotechnological approaches is currently limited by a lack of tissue culture - free transformation system. In this paper, we present the development of a gene transfer system using DNA imbibition [DI]; and/or silicone carbide fibers [SCF] treatments for gene delivery and seed-derived embryos as gene target. Plasmid DNA (pBI221.23), containing the selectable "hpt" gene for hygromycin resistance and the reporter "gus" gene, was delivered into mature wheat embryos via [DI] and/or [SCF] treatments. In a histochemical analysis of β-GUS activity, an average of 13.8 % of the mature embryos receiving the combined treatment [DI + SCF] showed gus expression, whereas only 3.2% and 0.4% showed gus expression following [SCF] and [DI] respectively. The mature embryos also showed more multi-site gus expression (4.6%) than those treated only with [SCF] (0.4%). The growth of the produced plantlets on a medium containing 45 µg/ml hygromycin B indicated that the transformation efficiency of the combined treatments [DI + SCF] was 0.8% in comparison to 0.1% for that of [SCF] treatment and no hygromycin resistant-plantlets were detected following [DI]. Stable transformation was confirmed through polymerase chain reaction "PCR" and Southern hybridization which indicated that a functional hpt gene had integrated into wheat chromosomal DNA.
4

Controversial aspect of using GFP as a marker for the production of transgenic cattle

84%
Duszewska A. M., Lipinski D., Piliszek A., Slomski R., Plawski A., Wojdan J., Gawron W., Juzwa W., Zeyland J., Wenta-Muchalska E., Reklewski Z.
Polish Journal of Veterinary Sciences
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2004
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tom 07
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nr 4
The objective of this study was to examine the feasibility of identification and selection of cattle embryos based on green fluorescence (GFP-positive) in order to obtain calves carrying an integrated transgene. The construct used (pbLGTNF-EGFP) contained the human tumor necrosis factor alpha (hTNFa) gene fused to the bovine betalactoglobulin promoter (bLG) in plasm id vector pCX-EGFP. In four experiments, 76 zygotes were injected; eight of them developed to the morulac/blastocysts stage of which only five were GFP positive (one of them 100%, one-50%, three- 25%). All of the GFP positive embryos were transferred to recipients. Two calves were born: one after transfer of the 100% GFP positive embryo and the other after transfer of one of the 25% GFP positive embryos. Both animals were healthy with normal weight when compared to two control calves. The integration of pbLGTNF-EGFP in the host genome could not be detected in either of the calves, suggesting that GFP is an unreliable marker for preimplantation screening of embryos.
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