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.
