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1

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

100%
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.
2

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

100%
Matejczyk M., Rosochacki S. J.
|
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.
3

Transgenic animals in agriculture and medicine

100%
Rosochacki S. J., Zwierzchowski L.
Biotechnologia
|
1999
|
issue 2
7-24
EN
One of the most important issues in the use of transgenic technology in animal breeding is production of meat. For that purpose growth enhancing DNA sequences are introduced to the genome of pigs, sheep, cattle, rabbits and fish. The structural elements of the introduced genes usually GH, GRF or IGF-I, which are combined with the regulatory elements. In the case of transgenic swine, the higher growth rate, and the lower fat content were achieved by using these gene constructs. However, a number of such swines were sterile and had some other pathological problems. Similar and even more serious problems, were noticed in the case of transgenic sheep up. To date, most transgenic livestock projects focused on enhancing growth in farm animals by overexpressing growth hormone have to pathological changes in transgenic farm animals; only in transgenic fish no such problems have been ecountered. Fast growing transgenic trouts, carps and salmons carrying the so called 'all fish gene constructs' could be used even right now as food. Another goal in transgenic livestock projects is manufacturing of biologicaly active human proteins in the mammary gland. Transgenic sheep, goats, pigs and cows which produce human pharmaceuticals in their milk have been obtained. Some of such proteins undergo clinical trials. In another arena, it is planned to modify the milk of ruminants in order to obtain better product, which will be used in the dairy industry. It is possible to introduce more copies of milk protein genes into the genome, 'improving' the existing genes, or to inhibit the expression of some genes - thus reducing or eliminating the production of unwanted proteins. Another way to modify milk composition is the so called 'humanisation' of cow's milk by changing the proportion of cow's to human proteins. So far, all these modifications in the area of milk proteins are done mostly on laboratory animals and the introduction of these possibilities to farm animals would be desirable. Except for fish, transgenic farm animals are relatively safe to the environment; the probability of transgenic animals surviving and reproducing out of farms is rather very low. On the contrary, introduction of highly productive farm animals (cows, pigs) to breeding would be profitable to the environment and the called 'transgenic bioreactors' would also cause decrease of chemical or pharmaceutical industry contamination to the environment. For consumers, the products obtained from transgenic animals should be safe. 'Transgenic' products, which normaly exist in the nature, are not more unsafe than their natural counterparts. There is a clear consumer demand for testing all new products which normaly do not exist in the nature, i.e. proteins with totaly new amino acid sequence, before their introducing into the market. However, this is also valid for all novel food products, not only those obtained by biotechnology.
4

Expression of microinjected reporter gene lacZ during first cleavages of rabbit embryos

61%
Rosochacki S. J., Strzelecka M., Kozikova L., Matejczyk M., Oblap R., Poloszynowicz J., Zwierzchowski L.
Folia Biologica
|
2002
|
vol. 50
|
issue 1-2
61-67
EN
The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta -galactosidase (beta -gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were coinjected with the scaffold attachment sequences - SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta -galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta -galactosidase expression. The percentage of transgenesis with pCMV-lacZ alone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.
5

Bovine mu-calpain (CAPN1) gene: new SNP within intron 14

61%
Juszczuk-Kubiak E., Sakowski T., Flisikowski K., Wicinska K., Oprzadek J., Rosochacki S. J.
Journal of Applied Genetics
|
2004
|
vol. 45
|
issue 4
457-460
EN
The calpain system orginally comprised molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. This proteolytic system plays a key role in the tenderisation process that occurs during post-mortem storage of meat under refrigerated conditioning. Their polymorphism is examined from the point of view of their effect on corresponding production traits. The calpain genes are investigated as potential candidate genes for a quantitative trait locus (QTL) affecting meat tenderness. In this study a new single nucleotide polymorphism (SNP) was found within intron 14 of the bovine CAPN1 gene, being transition CT at position 4685 nt (consensus sequence ? GenBank No. AF 248054), as this mutation creates a new FokI restriction site detected with PCR-RFLP analysis. This sequence fragment of the SNP position has already been deposited in the GenBank database under accession No. AY639597. The RFLP-FokI polymorphism was studied in 141 bulls of seven breeds, including the native Polish Red (PR, preserved), and Polish Black-and White (BW) breed. The frequency of alleles T and C varied between the breeds considered, the mean reaching 0.38 and 0.62, respectively. Associations between CAPN1/FokI gene polymorphism and meat production traits were studied in BW (n = 84) young bulls. In the animals of the TT genotype the lean share in valuable cuts (%) was found more favourable than in CC animals.
6

Noninvasive fluorescent screening of microinjected bovine embryos to predict transgene integration

61%
Rosochacki S. J., Kozikova L., Korwin-Kossakowski M., Matejczyk M., Poloszynowicz J., Duszewska A. M.
Folia Biologica
|
2003
|
vol. 51
|
issue 1-2
97-104
EN
Most transgenic domestic animals are generated by direct microinjection of DNA fragments into zygote pronuclei. It has generally been assumed that the majority of integration events should occur prior to the first round of chromosomal DNA replication. The aim of this study was to investigate the expression of GFP in bovine preimplantation embryos by using a gfp reporter gene consisting of chicken beta-actin promoter, the CMV-IE enhancer, gfp cDNA (EGFP) (732 bp) and rabbit beta-globin polyadenylation sequences. In five experiments 302 bovine zygotes were injected while 75 served as a control. The fluorescence intensity was detected at 72 and 168 h following fertilization in bovine embryos injected with 3 ng/mu l in experiments 1-3, and injected with 5 ng/mu l in experiments 4-5. Eight embryos were considered as expressing green fluorescence protein; 2 of them were 100% fluorescent after microinjection of a higher dose of the DNA; one was 75%, two - 50%, and three 25% transgenic. The mosaicism was assumed to be at 75%. The results indicated that the fluorescence could be analyzed at any time of bovine embryo development. It was therefore concluded, that chicken -actin promoter together with the CMV-IE enhancer would confer a strong expression of the gfp reporter gene in preimplantation bovine embryos. Therefore, using GFP that could be simply detected in live bovine (transgenic) embryos would be very promising in establishing transgenic lines of domestic animals producing in their fluids human therapeutic proteins.
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