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众多领域的理解。然而，尽管对鸟类的研究在发育、神经生物学和免疫学等领域做出了开创性的贡献，但由于基因操作工具的有限，包括制造转基因鸟类的能力，鸟类研究一直受到阻碍。在其他模式物种中，如小鼠、斑马鱼和果蝇，产生直接评估特定感兴趣基因作用的转基因动物要容易得多。一些最成功的转基因技术依赖于基因编辑、分离和修改干细胞，然后将它们移植到宿主体内。相比之下，鸟类在下蛋时胚胎的年龄较高，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。该项目的重点是斑马雀，这是一种发声学习的鸣禽，是研究人类言语和语言的神经和遗传基础最常用的动物模型。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，可能还包括任何产卵物种，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，并扩展到通常在科学和技术领域中代表性较低的社区。为了提供不仅对鸣禽研究社区有用，而且对一般鸟类研究人员也有用的基因操作工具和协议，研究人员如下：1)培育在普遍存在的启动子下表达基因组编辑酶Cas9的转基因斑雀；2)分离、培养和利用原始生殖细胞(PGC)以提高产生转基因斑雀的效率；以及3)开发高效的病毒载体来操作斑雀细胞中的基因。利用已建立的方法，将VSV假型慢病毒载体注射到新生的受精卵中，从而获得转基因斑马雀。当与CRISPR设计的引导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。为了扩大和创造许多转基因系，PGC的培养方法正在优化，并开发了在斑雀细胞中转染率更高的病毒载体，从而减少了创建新的转基因系所需的材料、时间和动物，并极大地促进了基因操作。该项目产生的工具和优化的方法也将影响鸟鸣生物学以外的领域的鸟类研究。PGC培养方法可以应用于其他鸟类，针对特定鸣禽组织优化的病毒载体在其他鸟类的类似细胞类型中也可能具有更高的转染率，Cas9-慢病毒构建物可用于产生其他表达Cas9的转基因禽类。

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.