Developing protein degradation technologies in avian systems

To understand the biological importance of proteins, the 'gold standard' is to modify them in a whole organism ('in vivo'). Methods for studying protein function in vivo are typically limited to genetic editing of DNA. Refinement of this to achieve conditional or targeted gene ablation requires knowledge and adaptation of tissue specific promoters.

This is laborious, has limited efficacy, and requires many animals. Therefore, new systems enabling rapid, reversible and targeted protein ablation in vivo are required. Recently, this has been achieved using tag-based degron strategies such as auxin inducible degron (AID) and PROTAC systems, which utilise small molecules to induce ubiquitin ligase mediated protein degradation.

These systems have great potential as an alternative and reversible method of gene/protein targeting, with utility across a range of contexts, cell types, and strategically relevant species. In this project, you will establish a tag-based protein degradation method in chicken, a powerful model to study vertebrate development, and implement it to investigate how migrating cells are influenced by different mechanical environments.

We have recently begun initial testing of both an AID and a BromoTag PROTAC system in chicken cells. You will directly compare the efficacy of these protein degradation systems in chicken cells, including primordial germ cells (PGCs), both in vitro and in vivo. After identifying the optimal system, you will use this to target YAP1, a key sensor and mediator of mechanical cues, which is vital for embryonic development.

Fusion target proteins, containing a degron tag and fluorescent protein to allow visualisation of native protein expression and localisation, will be generated through Crispr/Cas9 editing in PGCs. To test the functionality of target protein degradation you will assess the role of YAP1 in response to changes in mechanical environment both in vitro through modification of the culture conditions and also in vivo through reintroduction of edited PGCs into chick embryos.

In chickens, PGCs undergo multiple stages of migration involving movement through several different tissue types, each of which has distinct mechanical properties. By temporally ablating its function, you will determine the role of YAP1 in germ cell migration and maturation, and also address more generally how migrating cells respond to diverse mechanical stimuli.