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The transgenic chicken has great potential as a bioreactor for the production of valuable pharmaceutical proteins, notably in the oviduct/egg. Whereas conventional transgenic approaches have significant limitations in this species, an alternative approach employing primordial germ cells (PGCs), the progenitor cells to ova and spermatozoa, has now been successfully applied to the insertion of exogenous genes into birds. Recent developments in manipulating avian embryos make it possible to produce germline chimeras derived from transferred PGCs. In this review we describe the migration pathway of chicken PGCs during early development. We then summarize different methods for the isolation of PGCs and the diversity of techniques used to introduce genes into these cells. Finally, we describe an in vitro assay for testing tissue-specific vectors designed to express heterologous proteins in transgenic chickens.
The use of animals as a source of organs and tissues for xenotransplantation may overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans, directed against the swine Gal antigen present on the surface of xenograft donor cells, leads to the complement activation and an immediate xenograft rejection as a consequence of hyperacute reaction. In order to prevent a hyperacute rejection, it is possible to alter the swine genome with human genes modifying the set of the donor’s cell surface proteins. The aim of this study was to prepare a pCMVFut genetic construct and then introduce it into the swine genome in order to obtain transgenic pigs expressing human α1,2-fucosyltransferase and thereby avoid a humoral xenograft rejection. The pCMVFut gene construct containing the human gene encoding α1,2-fucosyltransferase enzyme under the human cytomegalovirus immediate early promoter was introduced by microinjection into a male pronucleus of a fertilized porcine oocyte. The screening procedure involved isolating genomic DNA from microsections of pigs’ ears, the amplification of two PCR fragments and the entire sequencing of positive samples. The mapping of the transgene was performed by fluorescence in situ hybridisation (FISH) and transgene expression, while its impact on the reduction of the Gal epitope level on the surface of pig cells was assessed by flow cytometry of primary cultured skin fibroblasts. The influence of the human complement was measured by testing the sensitivity of nontransgenic and transgenic cells to complement-mediated cytotoxicity upon exposure to human serum. As a result of this experiment, the founder male pig was obtained with the transgene mapping to chromosome 14q28. An RT-PCR analysis revealed the expression of the HT gene in different tissues of transgenic pigs. A flow cytometry analysis revealed a reduction in the level of epitop Gal on the cell surface of skin fibroblasts isolated from transgenic pigs. The complement-mediated cytotoxicity assay showed increased viability of transgenic cells in comparison with nontransgenic ones, which confirmed the protective influence of HT expression. In this study we demonstrated that the constitutive transgenic expression of human H-transferase (α1,2-fucosyltransferase) can decrease the amount of Galα1,3Gal (Gal epitope) on the surface of pig cells, which is consistent with the results of other researchers. The expression of α1,2-fucosyltransferase modified the cell surface carbohydrate phenotype of transgenic pig cells, resulting in the expression of the universally tolerated 0 blood group antigen (H antigen) and a subsequent reduction in the expression of Gal epitope, as evaluated by flow cytometry analysis. Apart from the principal data, the flow cytometry analysis revealed no significant differences between the Gal epitope level achieved by CMVFUT heterozygous boar founder TG 1154 and transgene homozygous pig 433 from the F2 generation. The flow cytometry results were confirmed by the cytotoxicity assay. We found no statistical difference in the survival rate between transgenic homozygous and heterozygous cells under the influence of 50% human serum with an active complement system. Both homozygous and heterozygous cells had the same level of lysis protection.
Successful experiments involving the production of transgenic mice by pronuclear microinjection are currently limited by low efficiency of random transgene integra­tion into the mouse genome. Furthermore, not all transgenic mice express integrated transgenes, or in other words are effective as functional transgenic mice expressing the desired product of the transgene, thus allowing accomplishment of the ultimate experimental goal - in vivo analysis of the function of the gene or gene network. The purpose of this review is to look at the current state of transgenic technology, utiliz­ing a pronuclear microinjection method as the most accepted way of gene transfer into the mouse genome.
Model organisms are essential to study the genetic basis of human diseases. Transgenic mammalian models, especially genetic knock-out mice have catalysed the progress in this area. To continue the advancement, further sophisticated and refined models are crucially needed to study the genetic basis and manifestations of numerous human diseases. Coinciding with the start of the new era of post-genomic research, new tools for establishment of transgenesis, such as nuclear transfer and gene targeting in somatic cells, have become available, offering a unique opportunity for the generation of transgenic animal models. The new technology provides important tools for comparative functional genomics to promote the interpretation and increase the practical value of the data generated in numerous mouse models. This paper discusses the state-of-the-art of the nuclear replacement technology and presents future perspectives.
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Enhanced food and water intake in renin transgenic rats

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In short term experiments angiotensin II (Ang II) is a potent stimulant of thirst, however it is not known whether prolonged activation of the renin-angiotensin system is associated with chronic alteration of water or food intake. Renin transgenic rats TGRmRen(2)27 (TGR) exhibit significant elevation of AngII in the brain regions involved in regulation of body fluid balance. The purpose of the present study was to find out whether TGR rats manifest also different water (WI) and food (FI) intake and renal excretory functions in comparison to their parent Sprague Dawley (SD) strain. To this end 24h WI and FI as well as urine excretion (Vu) and urinary outputs of solutes (Cosm), sodium (UNaV) and potassium (UKV) were compared under baseline conditions in 16 TGR and 15 SD rats having free access to water and food. In 15 TGR and 17 SD rats effect of 24h dehydration on water intake was investigated. Under baseline conditions TGR rats consumed significantly greater amount of food and water than SD rats. Vu, UNaV and UKV were not significantly different in both strains. Cumulative water intakes in SD and TGR rats subjected to 24h dehydration did not differ. The results reveal that under baseline conditions TGR rats manifest greater food and water intakes than SD rats whereas stimulation of thirst by water deprivation is similar in both strains. The results suggest that the ingestive behavior may be chronically altered by upregulation of the renin-angiotensin system.
Heat shock activates in somatic cells a set of genes encoding heat shock proteins which function as molecular chaperones. The basic mechanism by which these genes are activated is the interaction of the specific transcription factor HSF1 with a regula­tory DNA sequence called heat shock element (HSE). In higher eukaryotes HSF1 is present in unstressed cells as inactive monomers which, in response to cellular stress, aggregate into transcriptionally competent homotrimers. In the present paper we showed that the expression of a transgene encoding mutated constitutively active HSF1 placed under the control of a spermatocyte-specific promoter derived from the hst70 gene severely affects spermatogenesis. We found the testes of transgenic mice to be significantly smaller than those of wild-type males and histological analysis showed massive degeneration of the seminiferous epithelium. The lumen of tubules was devoid of spermatids and spermatozoa and using the TUNEL method we demonstrated a high rate of spermatocyte apoptosis. The molecular mechanism by which constitutively active HSF1 arrests spermatogenesis is not known so far. One can assume that HSF1 can either induce or repress so far unknown target genes involved in germ cell apoptosis.
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