dc.description.abstract
Transgenesis is a very effective tool to study the function of genes in vivo as it affords the best experimental control over particular cell types from the start of development. For popular animal models, such as mice or fruit flies, a variety of methods to generate transgenic animals are established and many transgenic strains are commercially available. In avian species, most research has been conducted on chickens and quails, but for songbirds, a pipeline to reliably produce transgenic individuals is still lacking. Songbirds offer a unique possibility to unravel questions concerning the neural basis of behavior, as many species are easy to keep and breed, exhibit a diversity of social behaviors including a rich repertoire of vocalizations used for communication. The use and production of many songbird vocalizations need to be learned during infancy, akin to speech learning in humans. Most research on the topic has been conducted on zebra finches (Taeniopygia guttata). A few publications describing the generation of transgenic zebra finches exist (Agate et al., 2009; Liu et al., 2015; Abe et al., 2015; Gessara et al., 2021), but have not enabled the scientific community of songbird researchers to create transgenic finches as a routine technique. It thus remains of great interest to investigate alternative methods or to improve on already published ones. In this thesis, two different methods for the generation of transgenic songbirds were tested. First, a method described in mice was tried to transfer to zebra finches involving microinjection and electroporation of testes. The difficulty was to overcome the different location of testes in birds and mice, e.g. inside versus outside of the body cavity respectively. Second, a previously published approach using progenitor cells of the germ line (PGC) was chosen (Gessara et al., 2021). This method was reported to lead to transgenic founders and offspring in a more efficient way than the first publication of transgenic finches, in which lentivirus was injected into the earliest stage of embryogenesis' (Agate et al., 2009). For the first approach, access to zebra finch gonads was achieved via laparotomy, and the manipulation of testes was conducted successfully without affecting male’s fertility. Different parameters were tested, and best conditions were determined by amount of reporter fluorescence in tissue sections of treated testes. Stable expression of the fluorescent reporter was achieved through stable integration of it by HypBase; suggesting that manipulation had long-term effect through integration of the transgene by transposition. The transgene could be detected in testes by PCR and slot blot genotyping as well as histology. Nevertheless, no transgenic offspring was identified yet. III For the second part of the study PGC extraction, culturing and transduction by lentivirus was implemented successfully. Similar levels in development of manipulated embryos as in Gessara et al., 2021 (45.5% vs. 56.3%) were generated. Hatching rates could not be compared as all tests were performed before 2/3 of embryonic development. Unfortunately, genotyping did not exhibit any transgenic founder or offspring among embryonic samples, which was the case for every hatchling in the previous study. In both approaches, difficulties with PCR genotyping (apparently due to contamination) were noted and consequently the method was switched to Slot blot, which lead to more reliable results. Here, a new method for transgenesis in zebra finches is presented that led to genetically modified testes of treated male zebra finches (through 'In vivo microinjection and electroporation of testes'). Unfortunately, this study was neither able to generate transgenic offspring by 'In vivo microinjection and electroporation of testes', used for the first time in a songbird, nor to repeat the outcome of Gessara’s PGC based approach. Still, both methods might be interesting for future studies as the Gessara publication had convincing more founder individuals than the classical 'Lentiviral injection into stage x embryos' approach. However, PGC heterogeneity (Jung et al., 2022) might be considered in future projects, that are PGCs based. Testes manipulation seems promising for songbird transgenesis after some refinements (e.g. optimized parameters, hemi-castration).
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