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

幼小的鸣禽通过模仿成年鸣禽来学习唱歌，使用的机制与婴儿学习说话时类似。因此，鸣禽是研究发声学习的分子、神经和生理基础的流行动物模型。然而，可靠的和常规的生殖系修改仍然缺乏。自2009年以来，只有三个实验室培育出转基因雀类，总共只有五个转基因品系。产生这些品系的效率很低，在1-10%之间。所有的斑雀转基因都是通过慢病毒注射到以原始生殖细胞(PGCs)为靶标的新鲜卵子(阶段X)的胚胎中产生的。这种方法产生的嵌合体F0首先需要与野生型杂交才能产生转基因F1，效率也很低(~13%)。第一只转基因鸡是在1986年由Salter和他的同事完成的，使用的方法与2009年第一只转基因斑马雀的方法相同。与此同时，已经开发出几种不同的、更有效的方法来产生转基因鸡和鹌鹑。因此，我们建议对在鸡和鹌鹑中建立的这些不同的方法进行比较，并采用它们来产生斑雀转基因株系。为了达到这个目的，我们建议比较慢病毒注射在不同胚胎阶段的结果，基于在胚胎后期产生转基因鸟更有效的结果。由于PGC培养尚未在雀类中建立，在我们的第二个目标中，我们专注于对精子或精原细胞的遗传操作，这些方法可以在一代中产生转基因鸡。为了达到这个目标，我们将操纵斑马雀精子，然后将其用于人工授精，正如库珀和他的同事最近所描述的那样。我们还将直接针对雄性斑马雀睾丸中的精原细胞。对于第三个目的，我们将选择在特定的鸣声系统神经元中产生表达通道视紫红质的斑马雀线来研究发声学习，即HVC中间神经元和X区中棘神经元。该项目的结果将是一个里程碑，使鸣禽成为真正的基因可修改的动物模型，研究发声学习以及社会和认知行为。

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.