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1

Selection systems in genetic engineering of plants

100%
Morag M., Lehmann P.
Biotechnologia
|
1999
|
issue 4
92-106
EN
Systems are required which allow selection of transformed cells. Genes that confer a selectable advantage to transformed cells are included in the transformation system. They include the use of antibiotics, herbicides or other compounds. In this review, we describe the systems developed for selective elimination of selectable marker genes from the transgenic plants.
2

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

100%
Stopa M., Dulak A., Jozkowicz A.
Biotechnologia
|
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.
3

Optimization of methods for the production of AAV vectors

100%
Jazwa A., Rutkowski A., Golda S., Rehhahn A., Jozkowicz A., Dulak J.
Biotechnologia
|
2007
|
issue 3
141-156
EN
One of the conditions of effective gene therapy is the choice of a proper gene carrier that will efficiently deliver the genetic material to the damaged tissue without causing deleterious side-effects. Adeno-associated viral vectors (AAV) have emerged as attractive tools for gene therapy, because of their broad tissue tropism, long-term transgene expression, and lack of human pathology. Nevertheless, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. In this article, we compare different methods for AAV production in order to optimize the conditions of AAV preparation to the scale and purity required for clinical and potential commercial applications.
4

Methods for the titration of AAV vectors

100%
Rutkowski A., Jazwa A., Popowa S., Jozkowicz A., Dulak J.
Biotechnologia
|
2007
|
issue 3
157-166
EN
Adeno-associated viral (AAV) vectors are promising tools for gene therapy. However, for trustworthy comparison of the results produced from different clinical trials, the exact amount of the used infectious vector particles must be known. We have produced AAV using a commercially available system and compared three common methods for the quantification of the number of produced vectors: ELISA, dot-blot and quantitavive PCR (qPCR). Although ELISA is a very reproducible and precise method, it is able only to determine the number of viral capsids in the vector preparation, also those which contain no genetic material and are therefore useless. With this method we established that we are able to produce ~ 6.5 x 1011 viral capsids/mL. Dot-blot assay determines the number of genomic particles in the vector preparation in a quite precise manner, but it is a very labor- and time-consuming method. qPCR is also a method determining the number of genomic particles. It is, however, much faster and simpler than the dot-blot assay. Both dot-blot and qPCR gave similar results (~ 4 x 1011 viral genomes/mL), which indicated only about 2/3 of the produced vectors containing genetic material. Our results show that qPCR is the most convenient and reliable method for quantification of AAV vectors and we believe it could be routinely used to titer the vectors prior to their usage in clinical trials.
5

Gfp gene as a fluorescence tool in gene expression analysis and biosensors construction

88%
Matejczyk M., Rosochacki S. J.
Biotechnologia
|
2007
|
issue 1
53-62
EN
When autofluorescent protein such as green fluorescent protein (GFP) is excited with light of a specific wavelength, it emits light of a longer wavelength without further addition of substrates. Gfp belongs to a family of reporter genes which provide easily detectable phenotypes to microbial cells and are, therefore, a valuable tool for the study of microorganisms in the environment, especially for analysis of processes such as microbe-plant interactions, biofilm formation, horizontal gene transfer (HGT) and gene expression analysis in different conditions (under influence of biological, physical and chemical factors), and bacterial biosensors costruction. In this paper, the main possibilities of gfp gene as a marker application in microbial ecology, gene expression analysis and biosensors development are presented.
6

The application of biological markers in the detection of environmental toxic compounds

88%
Rosochacki S. J., Matejczyk M.
Biotechnologia
|
2003
|
issue 1
208-215
EN
A biomarker, or molecular marker, or reporter gene is defined as a DNA sequence introduced into organisms. It confers a distinct genotype or phenotype to enable monitoring in a given environment. Molecular markers such as: LacZ (-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (-glucuronidase), gfp (green fluorescent protein), bla (-lactamase) and antibiotic or heavy metals resistance genes are widely used in genetically engineered (GEMs) microorganisms research. These genes are involved in the detection and enumeration of GEMs after their introduction into the environment. Molecular markers, especially lux and gfp, are widely used in the creation of whole-cell based biosensors which are commonly used for the examination of toxicity of environmental pollutants.
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