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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.
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