Transgenetic expression by cytoplasmic injection of plasmids and transposon-based constructs in mammalian embryos

Genetically modified animals play an important role as biomedical models and will have a considerable impact on agricultural production in the future. However, at the moment the use of transgenic animals is limited due to inefficient and expensive process. The aim of this study was to enhance the production of transgenic animals using cytoplasmic plasmid injection. In the first part of the work the expression of human telomerase in bovine blastocysts was investigated. Due to cytoplasmic injection of telomerase encoding plasmids the expression of human telomerase protein was induced, which resulted in significantly elongated telomeres. Since in the telomerase experiments the plasmids were not integrated in the genome, transgenesis was limited to approximately 7-14 days. Nevertheless, it did show how efficient injected plasmids are transcribed and translated. In the second part of the work the focus was to resolve the limitation of transient transgenesis. Therefore, cytoplasmic plasmid injection was combined with a non-autonomous transposon system. In a pilot project stable transgenic mice with a reporter transgen were produced. The transgen was integrated with specific transposition as a single copy. Subsequently, the transposon injection experiments were applied to pig zygotes. Transposon transgenic pigs were produced with high efficiency (42-57%). They show germline transmission and segregation of transposon transgene. It was possible to localise the transposon mediated transgene within the genome exactly and a Cre-recombinase exchange of the transgenic cassette against a functional transgene is possible. This approach allows a highly efficient production of transgene farm animals combined with a targeted integration of transgenes.