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1

Loop formation by the transgene WAP:6xHishGH in transgenic rabbit fibroblasts, revealed by fluorescence in situ hybridization to nuclear halos

100%
Michalak E., Lipinski D., Slomski R.
Journal of Applied Genetics
|
2006
|
vol. 47
|
issue 3
247-249
EN
Using fluorescence in situ hybridization (FISH) to somatic nuclear halos from transgenic rabbits WAP:6xHishGH, we present evidence for stability of transgenesis at the chromatin level. FISH performed on fibroblasts from a homozygous individual showed 2 independent loops from both chromosomes of pair 7. On a heterozygous individual, FISH detected a single loop. According to the concept of chromatin loops and their influence on gene expression, this shows that the human growth hormone transgene, which was actively expressed in mammary gland under the influence of the tissue-specific promoter, was inactive in examined skin fibroblasts.
2

The organ transplantation ? a challenge for biotechnology

100%
Jasinski A., Slomski R., Szalata M., Lipinski D.
Biotechnologia
|
2006
|
issue 1
7-28
EN
The use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. However, xenoreactive antibodies present in humans directed against swine Gal antigen on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. The graft of genetically modified organ of a swine would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. This review summarizes the clinical history and rationale for xenotransplantation, recent progress in understanding the physiologic, immunologic, and infectious obstacles to xenotransplantation and some of the strategies being pursued to overcome these dificulties.
3

Production of therapeutic proteins in the semen of transgenic animals

100%
Lipinski D., Juzwa W., Zeyland J., Slomski R.
Biotechnologia
|
2006
|
issue 1
44-52
EN
The expression of the recombinant proteins by transgenic animals represents an opportunity to achieve cost-effective, large-scale production of a wide variety of therapeutics. Among transgenic animal production systems, the transgenic mammary gland is the most advanced. However, the production of proteins in milk is limited by a relatively long interval from birth to first lactation encountered with domestic livestock, the discontinuous nature of the lactation cycle and the substantial time and material investments required to produce transgenic dairy animals. The semen of transgenic boar represents an alternative platform for the production of therapeutic proteins. The expression of such proteins in the male accessory glands, particularly in the seminal vesicle epithelium can be controlled by gene regulatory sequences specific to these tissues. In this review, we consider the possibility of using such regulatory sequences to drive the production of foreign proteins into the seminal fluids of transgenic animals. Application of this technology to pigs which can be ejaculated 2-3 times per week (200-300 ml per ejaculate), could lead to the annual production of several grams of recombinant protein.
4

Production of pigs used in xenotransplantation

64%
Jura J., Slomski R., Smorag Z., Gajda B., Wieczorek J., Lipinski D., Kalak R., Juzwa W., Zbyland J.
Biotechnologia
|
2006
|
issue 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.
5

Transgenic rabbit producing human growth hormone in milk

52%
Lipinski D., Jura J., Kalak R., Plawski A., Kala M., Szalata M., Jarmuz M., Korcz A., Slomska K., Gronek P., Smorag Z., Pienkowski M., Slomski R.
Journal of Applied Genetics
|
2003
|
vol. 44
|
issue 2
165-174
EN
The gene construct WAP(6xHisThr):hGH containing the entire human growth hormone gene (hGH) under the rat whey acidic protein (WAP) promoter regulating the expression in mammary glands of mammals was prepared. The 5? end of the gene was modified by the addition of a sequence encoding six histidine residues and a sequence recognized by thrombin. The gene construct was introduced by microinjection into the male pronucleus of a fertilized oocyte. The founder male rabbit was obtained with the transgene mapping to chromosome 7. The presence of the growth hormone was confirmed in samples of milk collected during the lactation of F1 generation females. The growth hormone can be easily purified by affinity chromatography and cleavage by thrombin to an active form.
6

Expressive gene structures of proper and modified genes

52%
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.
Biotechnologia
|
2003
|
issue 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.
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