Reviewer s comments on PhD thesis

Transcription

1 Reviewer s comments on PhD thesis The author of the PhD thesis: Mgr Inż. Izabela Kozłowska The title of PhD thesis: Behavior of chicken primordial germ cells injected into the bloodstream of recipient embryos Supervisor: prof. Dr. Hab. Marek Bednarczyk, Supervisor specialist: Dr. Inž. Pawel Lakota Reviewer: prof. Ing. Peter Chrenek, DrSc. Department of Biochemistry and Biotechnology, Faculty of Biotechnology and Food Science, Slovak University of Agriculture in Nitra, Slovak Republic The doctoral dissertation regards to primordial germ cells (PGCs) which are useful tool for the generation of transgenic birds and reconstruction of endangered avian breeds. The transgenic chicken has great potential as a bioreactor for the production of valuable pharmaceutical proteins, notably in the oviduct/egg. Whilst conventional transgenic approaches have significant limitations in this species, an alternative approach employing primordial germ cells 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.

2 Recently, gonadal PGCs (gpgcs) have been widely used to produce germline chimeric chickens because the collectable number of PGCs per embryo is the highest in gonads compared with that from other collection sites. The continuous maintenance of future generations in living organisms is preserved by germ cell development. Thus, germ cell research is important to advance infertility treatments and perform developmental studies. Elimination of endogenous germ cells has been widely used in germ cell transplantation studies (for clinical purposes) and germline chimera production (for research purposes). Several methods, including gamma-ray irradiation, X-ray irradiation, and bisulfan administration, have been developed to eliminate endogenous germ cells in different species. These methods primarily induce DNA damage in target cells, resulting in loss of all cellular mechanisms and ultimately cell destruction. Little is known about the cellular responses of PGCs after treatment with treosulfan as chemosterilizer with low toxicity. From these reasons, I think, that this topic is very actual. The thesis meets all requirements for such type of documents. It is clearly written and properly divided into 7 chapters. In the literature review, written on 13 pages, PhD student analyses current state of knowledge about the production method and use of transgenic chickens, primordial germ cells, as a tool for transgenic chicken production, morphological and biological characteristics of primordial germ cells, precise method for isolation and selection of primordial cells as a donor and recipient genotype. Totally 171 domestic and foreign literary sources were used in this paper, however, most of cited sources was originated from older years. The results of this thesis were logically divided into four parts. The purpose was to optimize some methods for isolation of primordial germ cells (PGC), selection of donor and recipients of primordial germ cells, sterilization of recipient embryos and to assess the impact of injected fluorochrome-labelled cells on development of embryos. The chapter Research Materials describes in vitro and in vivo experiments, starting from description and collection of biological material, list of laboratory equipment, chemicals, reagents and other materials used in the experiments. The experiments were focused on optimalization of the chicken primordial germ cell isolation from the gonads of 6-day old chicken embryos, impact of donor and recipient of cells on detection of exogenous primordial

3 germ cells in gonads of recipient embryos, sterilization method of recipient embryos and its effect on their survivability and development, as well as different methods of statistical analysis. Methodical approaches chosen for this study were adequate to the aims of the study. Obtained results and the novelty of the dissertation PhD thesis is focused on the actual problem of germline chimera generation by the PGC injection. The author obtained PGCs from the gonads using three different approaches. The highest number of PGCs, that did not form aggregates, was isolated by digestion of gonads with 0.25% trypsin EDTA. The author found a proper combination of the PGCs donor (ROSS 308) and the recipient (Green Legged Partridge), what allowed to obtain higher percentage of embryos with visible PKH26-labelled primary germ cells. In addition, this study has shown that treosulfan, as chemosterilizer, does not have a negative influence on the development of treated embryos. The results of the experiments are presented clearly, what gives a possibility to use the techniques examined in this study for further manipulations with avian PGC. PhD student presents obtained results in an appropriate form using tables, charts and figures. Comments and questions to the author General comments: 1. The abstract in English is missing. 2. I think that the order of the chapters should be different. For example, starting from chicken reproductive organs, characterization of chicken egg, chicken primordial germ cells, method for manipulations (isolation, culture conditions in vitro, ), transgenesis, chimera production, 3. Some methodological details and results are clearly presented in tables and pictures, but the numbering some of them is not correct and some titles are not in English (p. 81, 82, Tab. 24 and 25). 4. The incorrect description of Figures and Charts (Graphs), for example: Photos Fot., I think right is Fig., (not Fot.).

4 5. Some references are not correctly presented in the References, for example no. 157 (missing the year). 6. I appreciate the proper quality of Figures and Schemes that well illustrate the obtained results and thereby contribute to a better understanding of the experiments. Questions 1. You recovered primordial germ cells from gonads by partial digestion with 0.25% trypsin-edta. Did you or other researchers carry out any experiments with different concentration of trypsin-edta? 2. What is the reason of gonad incubation at 37.8 C, when the average body temperature of a chicken is around C? 3. What are practical and economical outcomes form the use of your results? In the "Discussion" author offers logical, highly detailed and substantive analysis of the own results in confrontation with those of other domestic and foreign authors. The section "Conclusions" brings detailed description of the obtained results in the exact order as the goals were raised: 1. Behavior of primordial germ cells depends on several factors (method of isolation, combination of donor recipient cells, etc ). 2. The highest number of morphologically normal primordial cells was obtained by incubating gonads digested with 0.25% trypsin-edta in PBS without calcium and magnesium. 3. The highest percentage of embryos with exogenous PKH26 labelled primordial germ cells was presented in the Ross 308 group. 4. Cytostatic treosulfan can be used for chemical sterilization of chicken embryos. 5. Cytostatic treosulfan does not significantly cause developmental abnormalities in treated embryos. Conclusion: Presenting the above opinion to the Board of the Faculty of Animal Breeding and Biology at the UTP University of Science and Technology in Bydgoszcz, I confirm that

5 evaluated PhD thesis Behavior of chicken primordial germ cells injected into the bloodstream of recipient embryo corresponds fully to the defined requirements, specified by the art. 26 ustawy z dnia r. o stopniach naukowych i tytule naukowym oraz o stopniach i tytule w zakresie sztuki, z późniejszymi zmianami i Ustawą Prawo o szkolnictwie wyższym. On this basis, I recommend the admission of MSc. Izabela Kozlowska to the next stage of a PhD proceeding. At the same time, in the recognition of the enormous work done during the objectives pursued, using a wide panel of interdisciplinary techniques and taking into account the nature and significance of the results for the development of our knowledge about the behavior of primordial germ cells, I put the request to award the prize to the PhD thesis. In Nitra, August, 31 th, 2015 prof. Ing. Peter Chrenek, DrSc.