|
|
Zebrafish as a model to study the glutamate receptor mediated excitatory neurotrasmission
Chumchal, Lukáš ; Balík, Aleš (advisor) ; Pangrácová, Marie (referee)
Glutamate is the most prominent excitatory neurotransmitter in the vertebrate brain and is used by most synaptic connections in the cortex. Signal transduction on these neurons is mediated by ionotropic glutamate receptors, including the NMDA receptor family. With the development of molecular biological methods and the advent of genomics, genetic changes found in ionotropic glutamate receptors were tested, as well as substances that modulate their activity. Since a large number of genetic changes found, rodents have ceased to be a sufficiently robust system for some, for example, behavioural studies. In these types of studies, the model organism Danio rerio could replace rodents. The use of this model organism could thus expand knowledge about the evolution and physiology of glutamate receptors. This work aims to summarize the current knowledge about the use of Dania rerio in the research of glutamate receptors, especially NMDA type in the central nervous system. This work also focuses on the description of specific behavioural tests available for the analysis of these receptors.
|
|
|
Role of Rnf207b in zebrafish hematopoiesis
Vondráková, Zuzana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...
|
|
|
Manipulace zárodečných buněk jako nástroj pro management a produkci izogenních linií u ryb
FRANĚK, Roman
Isogenic lines in fish represent a fundamental approach to control the genetic background of experimental animals. All individuals from a given isogenic line share the same genotype. So far, isogenic fish lines have been produced only by repeated uniparental inheritance - androgenesis and gynogenesis. Homozygous progeny is produced in the first generation of uniparental inheritance, and each homozygous individual produces a different isogenic line after second generation of uniparental inheritance. Despite optimized procedures for inducing uniparental inheritance, isogenic lines have been successfully produced in only a few species of fish. Doubled haploids after first uniparental inheritance have affected fitness as well as reproductive performance. Long-term maintenance is considerably problematic even when isogenic line is established already, due to low viability and poor reproductive characteristics. The situation is further complicated by the fact that isogenic lines are usually naturally monosex, thus uniparental inheritance must be re-used for further reproduction, or sex reversal needs to be applied in part of isogenic line. Several types of germ cell manipulation were performed in presented thesis. Protocols for cryopreservation of spermatogonia and oogonia have been developed and optimized to maximize post-thaw viability. The physiological activity of cryopreserved cells was confirmed by transplantation into a surrogate host. Cryopreserved and subsequently transplanted cells retained colonization activity comparable to non-frozen control germ cells. More importantly, male germ cells were able to transdifferentiate from oogonia. The success of transplantation was confirmed by detection of expression of genes associated with gametogenesis in carp by RT-PCR. In the next study, the results of cryopreservation experiments were followed, where sterile goldfish was identified as a suitable host for homozygous carp cells. Germ cells obtained from several homozygous individuals were individually transplanted into sterile goldfish. This procedure has a potential to increase the chance of producing a viable gamete for isogenic line production. Germ cells from homozygotes with affected gametogenesis can be transferred to fully viable recipients, thereby increasing the efficiency of isogenic line production overall. In addition, the use of a goldfish as a surrogate parent will ensure that part of the germline chimeras will be male and female, thus isogenic gametes of both sexes can be obtained and no further intervention for further reproduction of the isogenic line. The suitability of triploid zebrafish, which can potentially be used as recipients for cells from homozygotes to produce isogenic lines, has been confirmed for zebrafish. Spermatogonia and oogonia from diploid donors were transplanted into artificially induced triploid larvae. Donor-derived sperm was were obtained upon maturation of triploid recipients. Transplanted oogonia transdifferentiated into spermatogonia and spermatozoa with female sex chromosomes have been produced, which may be of interesting for further studies of sex determination in zebrafish. A new germline transfer technique has been developed using striped embryos. Donor cells were transplanted from the blastula stage to the swim-up larvae. With this approach, undifferentiated primordial germ cells were able to colonize the genital groove and initiate gametogenesis. After reaching sexual maturity, germline chimeras were obtained with gametes and viable progeny. Although the overall efficacy of this method was lower compared to other transplantation methods, this study may be of relevance for germline rescue in poorly viable embryos or lethal mutants.
|
|
|
Optimization of the rearing of the species Danio rerio (Hamilton, 1822)
Gottwald, Milan ; Kalous, Lukáš (advisor) ; Martin, Martin (referee)
The diploma thesis summarizes the experimental method for the rearing of zebrafish Danio rerio (Hamilton, 1822) screed. The work of figuring out which of the methods of rearing fry in the transitional period of the ontogenetic development is best for the survival rate and growth of the standard length of the surveyed individuals.
The test was carried out on two basic lines of zebrafish Wild Type and Casper. At the beginning of the test were created four groups divided by feeding mode. The control group was fed once a day with pellet feed GEMMA Micro 75 and one day from the 10th day after fertilization the fed with nauplii stages of brine shrimps Artemia salina (Linnaeus, 1758). The second group was fed only once a day with pellet feed GEMMA Micro 75. The last two groups were fed five times a day saltwater rotifer species Brachionus plicatilis (Müller, 1786), and only one of them and the other was still three times daily nourished with nauplii stages of brine shrimps. For each group of four representative have been created initially, thirty tanks of fish at the age of five days after fertilization.
The results of this method have shown that the methods they use to feed the rotifers, did not have a statistically significant impact on the survival rate of individuals. In the growth of this method has proven to be statistically significant result in the group where the fed in combination with brine shrimps, where to achieve the average standard size of juvenile fish 16.02 +/- 0.80 mm for the Wild type and 17.39 +/- 0.81 mm for Casper, compared to the control group, that has been the standard length 11.63 +/- 0.64 mm for the Wild Type and 9.54 +/- 0.56 mm for Casper.
This method has great potential and breeding is therefore necessary to further develop this method and to adapt it to individual zebrafish facilities.
|
guest ::
