Optimization and testing of new methods for the generation of zebra finch transgenics

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Young songbirds learn to sing by imitating adult conspecifics using similar mechanisms as infants do when learning to speak. As a result songbirds are a popular animal model to study the molecular, neural and physiological underpinnings of vocal production learning. However, reliable and routine germline modifications are still lacking. Since 2009 only three laboratories have produced transgenic finches, in total only five transgenic lines. Efficiency to generate these lines was low, between 1-10%. All zebra finch transgenics were produced by lentiviral injection in embryos of freshly laid eggs (Stage X) targeting primordial germ cells (PGCs). This method generates a chimeric F0 that first needs to be crossed with a wild type to generate a transgenic F1, also with low efficiency (~13%). The first chicken transgenic was done in 1986 by Salter and colleagues, using the the same method as done for the first transgenic zebra finch in 2009. In the meantime, several different and more efficient methods to generate transgenic chickens and quails have been developed. Therefore we propose to compare these different methods established in chicken and quail and adapt them to generate zebra finch transgenic lines. Towards this aim, we propose to compare lentiviral injections at different embryonic stages, based on results from quails in which later embryonic stages were more efficient in generating transgenic birds. Since PGC culture is not established in finches yet, in our second aim we focus on genetic manipulations of sperm or spermatogonial cells, methods that can generate transgenic chickens in one generation. Towards this aim, we will manipulate zebra finch sperm that will be then used for artificial inseminations as described by Cooper and colleagues recently. We will also directly target spermatogonial cells in the testis of male zebra finches. For the third aim we will choose the best approach to generate zebra finch lines expressing Channel rhodopsins in specific song system neurons to study vocal learning, i.e. HVC interneurons and Area X medium spiny neurons. Results of this project will be a milestone towards making songbirds a genuine genetically modifyable animal model to study vocal production learning, as well as social and cognitive behavior.