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1

Cryoconservation of mammalian embryos and oocytes

100%

Gajda B. , Smorag Z.

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nr 2

10-33

EN

This paper presents current methods of embryo and oocyte cryoconservation in the following species: mice, rabbits, sheep and goats, pigs, horses and cows. Both the freezing and vitrification methods are discussed with special emphasis on the major factors affecting the efficiency of these two methods.

2

Vitrification of mammalian oocytes and embryos

100%

Smorag Z. , Gajda B.

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nr 3

68-83

EN

Vitrification is a new approach to oocyte and embryo cryoconservation.It consists in the solidification of a solution caused by draastic increase in viscosity during cooling and not by crystalization.The application of this approach to cryoconservation of oocytes and embryos of different species depends upon the development of proper procedures and non-toxic media.From the technical point of view, the vitrification method is simple and relatively easily applicable under field conditions.The authors review the current procedures applied to oocytes and embryos of laboratory and farm animals.

3

Ethical aspects of animal biotechnology

100%

Smorag Z.

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nr 3

7-12

4

Effect of protein source, witamin E and phenazine ethosulfate on developmental competence and quality of porcine embryos cultured in vitro

80%

Gajda B. , Baryla M. , Smorag Z.

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nr 1-2

57-63

EN

Effects of fetal calf serum (FCS) or bovine serum albumin (BSA), with or without vitamin E (vit. E) or phenazine ethosulfate (PES) supplementation on developmental competence and quality of cultured porcine embryos were examined. The experiment was done on zygotes and 2-cell embryos obtained from superovulated gilts. Morphologically normal zygotes were cultured in vitro in NCSU-23 medium supplemented with: experiment 1-0.004 g/ml BSA, 10% FCS, protein-free (control); experiment 2-0 (control), 25, 50 or 100 M vit. E; experiment 3-0 (control), 0.025, 0.05 or 0.075 M PES. Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL staining. Presence of BSA in culture medium increased significantly morula and blastocysts production as compared to FCS (P<0.001) and protein-free group (P<0.05 and P<0.001, respectively). The blastocysts cultured in protein-free medium had higher average number of apoptotic nuclei and DNA fragmented nucleus index as compared to the BSA (P<0.05 and P<0.01, respectively) and FCS (P<0.5) group. Supplementation in culture medium of 100 M vit. E increased blastocyst production as compared to control and 50 M vit-E (P<0.05). Both the number of cells per and percentage of TUNEL positive nuclei per blastocyst were slightly lower in PES treated than control groups.

5

Production of pigs used in xenotransplantation

51%

Jura J. , Slomski R. , Smorag Z. , Gajda B. , Wieczorek J. , Lipinski D. , Kalak R. , Juzwa W. , Zbyland J.

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nr 1

151-158

EN

There are four main trends in the use of transgenic animals. One of the most interesting is the use of transgenic animals as the donors of tissue or organs suitable for transplantation in human ? xenotransplantation. This field of research has been undergoing intensive and increasing study during the past few years, and some encouraging progress is being made. A pig has been identified as the most suitable donor animal. The aim of the presented experiment was to produce transgenic pigs which tissues and organs could be used for the xenotransplantation needs. To target the goal, a competitive gene construct coding the same substrate as the endogenous enzyme of organ donor was introduced. In effect, transgenic boar was produced with confirmed integration of human a1,2 fucosyltransferase gene. Also, F1 generation of transgenic pigs was generated to preserve ongoing needs of preliminary research of xenotransplantation project.

6

Expressive gene structures of proper and modified genes

41%

Lipinski D. , Szalata M. , Kalak R. , Plawski A. , Nuc K. , Kala M. , Juzwa W. , Slomska K. , Gronek P. , Jura J. , Smorag Z. , Pienkowski M. , Slomski R.

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2003

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nr 1

48-73

EN

The genetic construct WAP 6xHisHGH containing the gene encoding human growth hormone (hGH) and WAP promoter expressed in mammary gland of animals was prepared. The 5? end of the gene was modified by the addition of sequence encoding six histidine residues and the sequence recognized by thrombin. In this way, the growth hormone can be easily purified by affinity chromatography and cleaved with thrombin to an active form. In the next step, the genetic construct was introduced by microinjection into male pronuclei of fertilized oocytes. Transgene was detected in male rabbit of F0 generation (number 61). Twelve offspring of founder rabbit of generation F1 indicated transgene sequences. The presence of growth hormone was revealed in the samples of milk accumulated during the lactation of females of F1 generation. The genetic constructs containing chain 1 and chain 2 of Feld1, and the major allergen produced by cat (Fedlis domesticus) were prepared. Both genes were inactivated by introduction into the sequences a positive selectable marker aminoglycoside phosphotransferase (resistant to neomycin). Outside the region of homology to Feld1 chain 1 and chain 2 genes, the negative selectable marker ? thymidine kinase gene was introduced. The genetic constructs pNTKFd1 and pNTKFd2 can be used in further experiments involving the inactivation of Feld1 genes in cat cells. Both genes were modified by site-directed mutagenesis using megastarter with Stop codon for premature termination of translation. The presence of mutation was confirmed by sequencing. The genetic constructs with human hGH gene and cat Feld1 gene were introduced into the bovine and cat fetal fibroblasts respectively in co-transfection with plasmid pGT-N29 containing positive selectable marker by lipofection, precipitation and electroinjection methods. After the selection, surviving cells were subjected to further molecular analysis. The stabile incorporation of the genetic constructs WAP 6xHisHGH and WAPHGH into the genome were observed.