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

斑胸草雀转基因产生新方法的优化和测试

• 批准号: 429335898
• 负责人: Professorin Constance Scharff, Ph.D.
• 金额: --
• 依托单位: Arbeitsgruppe Verhaltensbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/429335898?language=en
• 关键词: Optimization; testing; new; methods; generation

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.

幼小的鸣禽通过模仿成年同类来学习唱歌，其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，仍然缺乏可靠和常规的种系修饰。 自2009年以来，只有三个实验室培育出转基因雀，总共只有五个转基因品系。 生成这些线的效率很低，在 1-10% 之间。所有斑胸草雀转基因都是通过在新产卵的胚胎（X 阶段）中注射慢病毒来靶向原始生殖细胞（PGC）而产生的。该方法生成嵌合 F0，首先需要与野生型杂交才能生成转基因 F1，效率也较低（约 13%）。 1986 年，Salter 及其同事使用与 2009 年首次转基因斑胸草雀相同的方法，完成了第一只转基因鸡。与此同时，已经开发了几种不同且更有效的方法来产生转基因鸡和鹌鹑。因此，我们建议比较在鸡和鹌鹑中建立的这些不同方法，并采用它们来产生斑胸草雀转基因品系。为了实现这一目标，我们建议根据鹌鹑的结果比较不同胚胎阶段的慢病毒注射，其中后期胚胎阶段在产生转基因鸟类方面更有效。由于 PGC 培养尚未在雀类中建立，因此我们的第二个目标是对精子或精原细胞进行基因操作，这种方法可以在一代内产生转基因鸡。为了实现这一目标，我们将操纵斑胸草雀的精子，然后将其用于人工授精，正如库珀及其同事最近描述的那样。我们还将直接靶向雄性斑胸草雀睾丸中的精原细胞。对于第三个目标，我们将选择最佳方法来生成在特定鸣叫系统神经元中表达通道视紫红质的斑胸草雀系，以研究声音学习，即 HVC 中间神经元和 X 区中型多刺神经元。该项目的结果将是一个里程碑，使鸣禽成为真正的基因改造动物模型，用于研究发声学习以及社会和认知行为。