ImPaqT, a Golden Gate-based immunological toolkit for zebrafish transgenesis

Transgenic animals play an essential role in many aspects of zebrafish research. Here, we have developed ImPaqT (Immunological Toolkit for PaqCI-based Golden Gate Assembly of Tol2 Transgenes), a new Tol2-based transgenesis system that utilizes Golden Gate assembly to facilitate the production of transgenic zebrafish lines. This system allows for rapid assembly of multiple fragments into a single transgene, facile swapping of individual sequences to generate new transgenes, and an easy cloning workflow to incorporate new genetic elements into the existing kit. Within this framework, we have generated reagents to enable gene expression within different cell types, an array of best-in-class fluorescent proteins to visualize cell populations and transgenes, as well as tools to simplify genetic manipulation, purification, and ablation of targeted cells. Unlike many recombination-based systems, our approach is also expandable, allowing the incorporation of complex designs such as multifragment promoters within the established modular framework of ImPaqT. We have demonstrated the function of our system by generating various transgenic immune reporter lines. While we focused on the immune system as an emerging area of study within zebrafish research, ImPaqT can be broadly adapted to the construction of almost any zebrafish transgene, offering new tools for the zebrafish community.