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Nützel, Friedrich (2005): Relative Quantifizierung vaskulogenese- und angiogeneseassoziierter Wachstumsfaktoren der frühen Embryonalentwicklung des Huhns (Gallus gallus domesticus) mittels der real-time RT-PCR. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Aim of the study was the description of the transcriptional profile of different vasculogenic, angiogenic and haematogenic growth factors (Flk-1, Flt-1, VEGF, c-kit, SCF, VE-Cadherin, FGF-2, Ang-1, BMP2, BMP4) in the tissues of early vessel- and blood cell formation of the chick embryo. For this purpose the quantitative real-time RT-PCR was used, with which the amount of defined transcripts in previously processed tissue samples can be determined. In the course of the first seven days (E0 – E7) of embryologic development tissue samples of the two extraembryonic haemangiopoetic organs, the yolk sac and the allantois/chorioallantois, as well as of the embryo itself were taken and the mRNA-quantity of the numbered factors determined as mentioned above. By this means a comparison of the transcriptional levels in the different samples was achieved and enabled relative quantification. So the transcriptional activity and the approximate time course of gene expression of the investigated growth factors in the first third of chick development could be determined. It was shown that the transcriptional activity of the genes of interest was generally higher in the allantois/chorioallantois compared to the yolk sac. This imbalance even grew constantly from embryonic age E4 to E7. Thus in the allantois/chorioallantois a more intense vasculogenesis/angiogenesis compared to the yolk sac was taking place and according to that the higher amount of VE-Cadherin suggested a higher fraction of endothelial cells in the tissues of the allantois/chorioallantois. The target genes Flk-1, Flt-1_cons, VEGF und VE-Cadherin showed only small fluctuations of the cellular mRNA content over the course of the observed development, i.e. they were transcribed with a quite stable intensity. For the target genes Flt-1_ms, c-kit, SCF, BMP2 und BMP4 minor increases as well as minor decreases of the cellular mRNA content were observed, which varied from one day of embryologic development to the next with a factor in the range of 0,2 to 5. Differential transcriptional intensities higher than that were only observed for FGF-2 and Ang-1. For FGF-2 in the yolk sac, respectively Ang-1 in the allantois/chorioallantois, an approximate sevenfold increase of the transcriptional level from E4 to E5 could be shown. This may underline the importance of the correlated proteins for the ontogenesis of these organs at that point of time. In the embryo from E1 to E2 an approximate 40fold increase of the cellular mRNA content of Ang-1 was observed, which was followed by an about 20fold increase up to E3. From E1 to E3 these increases summed up to an almost 1000fold increase, which was by far the strongest regulational activity observed in this study. In the yolk sac a similarly high amount of Ang-1 mRNA was detected, suggesting an effective translation, great importance of the correlating angiogenic proteins for the maturation of the primary capillary plexuses in the embryo as well as in the yolk sac after three days of extrauterine development can be postulated. The determination of the individual amplification efficiency of each reaction of the real-time RT-PCR with the help of computer-based linear regression analysis resulted for the first cycles with detectable amplification and according increase in fluorescence-intensity in a multiplication to far over the double up to the fourfold RFU-value from one cycle to the next. This contradicts the theoretical maximum of an exponential amplification to the base of two and could be the result of a quantitative imbalance in the limited matrix-molecules compared to the other, prominent at the beginning of the reaction in extense present, PCR-educts. An intense observation of the reaction kinetics of exponential phase PCR-amplification seems helpful for an even more exact nucleotide-quantification.