IOS EDGE: Developing gene manipulation tools and resources for a vocal learning species

在过去的四十年里，基因操作技术的进步极大地提高了我们对生物学许多领域的理解。然而，尽管鸟类研究对发育、神经生物学和免疫学等领域做出了开创性的贡献，但鸟类研究一直受到基因操作工具有限的阻碍，包括制造转基因鸟类的能力。在其他模型物种中，如小鼠、斑马鱼和果蝇，更容易产生直接评估特定目标基因作用的转基因动物。一些最成功的转基因技术依赖于基因编辑、分离和修饰干细胞，然后将它们移植到宿主体内。相比之下，在鸟类中，产卵时胚胎的年龄很大，缺乏有效的病毒载体工具，极大地限制了基因操作的努力。为了解决这些局限性，研究人员开发和改进了鸟类的基因操作和干细胞技术。该项目的重点是斑胸草雀，这是一种学习发声的鸣禽，是研究人类语音和语言的神经和遗传基础的最常用动物模型。由于研究中常用的其他动物模型没有发声学习，这是第一次为发声学习者物种开发有效的基因操作方法。其他受益者包括所有鸟类研究和可能的任何产卵物种，其中许多用于解决生物学不同领域的问题。该项目还为高中生、本科生和研究生提供了研究机会，并向科学和技术领域通常代表性不足的社区提供了外展机会。为了提供不仅对鸣禽研究界有用的基因操作工具和协议，而且对一般鸟类研究人员也有用，研究人员是：1）产生在普遍存在的启动子下表达基因组编辑酶Cas9的转基因斑胸草雀; 2）分离、培养和利用原始生殖细胞（PGCs）以提高产生转基因斑胸草雀的效率;以及3）开发用于在斑胸草雀细胞中操纵基因的有效病毒载体。使用已建立的方法，通过将VSV假型慢病毒载体注射到新鲜产下的受精卵中来制备转基因斑胸草雀。当与CRISPR设计的指导RNA结合时，这些转基因鸣禽能够在各种组织和细胞类型中实现基因编辑能力。为了扩大规模并创建许多转基因品系，正在优化PGC培养方法，并开发在斑胸草雀细胞中具有更高转染率的病毒载体，从而减少创建新的转基因品系所需的材料，时间和动物，以及大大促进基因操作。该项目所产生的工具和优化方法也将影响鸟鸣生物学以外领域的鸟类研究。PGC培养方案可以应用于其他鸟类物种，针对特定鸣禽组织优化的病毒载体也可以在其他鸟类物种的类似细胞类型中具有更高的转染率，并且Cas9-慢病毒构建体可以用于产生表达Cas9的转基因鸟类的其他菌株。

Over the past four decades, advances in gene manipulation technologies have dramatically improved our understanding of numerous fields in biology. However, although studies in birds have made seminal contributions to fields such as development, neurobiology, and immunology, bird research has been hindered by the limited availability of gene manipulation tools, including the ability to make transgenic birds. In other model species, such as mice, zebrafish, and fruit flies, it is much easier to generate transgenic animals that directly assess the role of a particular gene of interest. Some of the most successful transgenic technologies rely on gene-editing and isolating and modifying stem cells, and then transplanting them into hosts. In contrast, in birds, the advanced age of embryos at the time eggs are laid, and the lack of efficient viral vector tools has greatly limited gene manipulation efforts. To address these limitations, the investigators develop and improve gene manipulation and stem cell technologies in birds. The project focuses on the zebra finch, a vocal learning songbird that is the most commonly used animal model to study the neural and genetic basis of human speech and language. Since other animal models commonly used in research do not have vocal learning, this is the first time that efficient methods for gene manipulation are being developed for a vocal learner species. Other beneficiaries include all avian research and possibly any egg-laying species, many of which are used to address questions in diverse areas of biology. The project also provides research opportunities for high school, undergraduate, and graduate students and outreach to communities typically underrepresented in science and technology fields.To provide gene manipulation tools and protocols that will be useful not only to the songbird research community, but also to avian researchers in general, the investigators are: 1) generating transgenic zebra finches that express the genome-editing enzyme Cas9 under a ubiquitous promoter; 2) isolating, culturing, and utilizing primordial germ cells (PGCs) to improve the efficiency of generating transgenic zebra finches; and 3) developing efficient viral vectors for manipulating genes in zebra finch cells. Using established methods transgenic zebra finches are made by injecting VSV-pseudotyped lentiviral vectors into freshly-laid fertilized eggs. When combined with CRISPR-designed guide RNAs, these transgenic songbirds enable gene-editing capabilities in a variety of tissues and cell types. To scale up and create many transgenic lines, PGC culture methods are being optimized and viral vectors with higher transfection rates in zebra finch cells developed, thus reducing the materials, time, and animals needed to create a new transgenic line, as well as greatly facilitating gene manipulations. The tools and optimized methods generated by this project will also impact avian research in fields other than birdsong biology. PGC culturing protocols can be applied to other avian species, viral vectors optimized for specific songbird tissues may also have higher transfection rates in analogous cell types of other avian species, and the Cas9-lentiviral construct could be used to generate other strains of Cas9-expressing transgenic birds.