ImPaqT - A Golden Gate-based Toolkit for Zebrafish Transgenesis
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Curated by eLife
eLife Assessment
This study introduces a useful toolkit for zebrafish transgenesis, significantly enhancing the flexibility and efficiency of transgene generation for immunological applications. The authors provide supporting evidence through well-designed experiments, demonstrating the toolkit's utility in generating diverse and functional transgenic lines. While the findings are solid, additional functional validation and broader comparisons to existing systems would strengthen the overall evidence base and ensure broader relevance to the zebrafish field, thereby increasing the significance of the study.
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Abstract
Transgenic animals continue to play an essential role in many aspects of zebrafish research, including the development of disease models. The most widely used system for zebrafish transgenesis is the Tol2 transposon system. Here, we have developed ImPaqT ( Im munological toolkit for Paq CI-based Golden Gate Assembly of Tol2 T ransgenes), 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 toolkit framework, we have generated a number of reagents to enable gene expression within immune and non-immune 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 recombination-based systems, the Golden Gate approach is also expandable, allowing the incorporation of complex designs such as multi-fragment promoters within the established modular framework of ImPaqT. Here, we demonstrate the function of this new system by generating a number of novel transgenic immune reporter lines. While our toolkit is focused on the immune system as an emerging area of study within zebrafish research, the ImPaqT approach can be broadly adapted to the construction of almost any zebrafish transgene, offering new tools for the generation of transgenes within the zebrafish community.
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eLife Assessment
This study introduces a useful toolkit for zebrafish transgenesis, significantly enhancing the flexibility and efficiency of transgene generation for immunological applications. The authors provide supporting evidence through well-designed experiments, demonstrating the toolkit's utility in generating diverse and functional transgenic lines. While the findings are solid, additional functional validation and broader comparisons to existing systems would strengthen the overall evidence base and ensure broader relevance to the zebrafish field, thereby increasing the significance of the study.
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Reviewer #1 (Public review):
Summary:
The authors introduce ImPaqT, a modular toolkit for zebrafish transgenesis, utilizing the Golden Gate cloning approach with the rare-cutting enzyme PaqCI. The toolkit is designed to streamline the construction of transgenes with broad applications, particularly for immunological studies. By providing a versatile platform, the study aims to address limitations in generating plasmids for zebrafish transgenesis.
Strengths:
The ImPaqT toolkit offers a modular method for constructing transgenes tailored to specific research needs. By employing Golden Gate cloning, the system simplifies the assembly process, allowing seamless integration of multiple genetic elements while maintaining scalability for complex designs. The toolkit's utility is evident from its inclusion of a diverse range of promoters, …
Reviewer #1 (Public review):
Summary:
The authors introduce ImPaqT, a modular toolkit for zebrafish transgenesis, utilizing the Golden Gate cloning approach with the rare-cutting enzyme PaqCI. The toolkit is designed to streamline the construction of transgenes with broad applications, particularly for immunological studies. By providing a versatile platform, the study aims to address limitations in generating plasmids for zebrafish transgenesis.
Strengths:
The ImPaqT toolkit offers a modular method for constructing transgenes tailored to specific research needs. By employing Golden Gate cloning, the system simplifies the assembly process, allowing seamless integration of multiple genetic elements while maintaining scalability for complex designs. The toolkit's utility is evident from its inclusion of a diverse range of promoters, genetic tools, and fluorescent markers, which cater to both immunological and general zebrafish research needs. Furthermore, the modular design ensures expandability, enabling researchers to customize constructs for diverse experimental designs. The validation provided in the manuscript is solid, demonstrating the successful generation of several functional transgenic lines. These examples highlight the toolkit's efficacy, particularly for immune-focused applications.
Weaknesses:
While the toolkit's technical capabilities are well-demonstrated, there are several areas where additional validation and examples could enhance its impact. One limitation is the lack of data showing whether the toolkit can be directly used for rapid cloning and testing of enhancers or promoters, particularly cloning them directly from PCR using PaqCI overhangs without needing an entry vector. Similarly, the feasibility of cloning genes directly from PCR products into the system is not demonstrated, which would significantly increase the utility for researchers working with genomic elements.
The authors discuss potential applications such as using the toolkit for tissue-specific knockout applications by assembling CRISPR/Cas9 gRNA constructs. However, they do not demonstrate the cloning of short fragments, such as gRNA sequences downstream of a U6 promoter, which would be an important proof-of-concept to validate these applications. Furthermore, while the manuscript focuses on macrophage-specific promoters, the widely used mpeg1.1 promoter is not included or tested, which limits the toolkit's appeal for researchers studying macrophages and microglia.
Another potential limitation is the handling of sequences containing PaqCI recognition sites. Although the authors discuss domestication to remove these sites, a demonstration of cloning strategies for such cases or alternative methods to address these challenges would provide practical guidance for users.
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Reviewer #2 (Public review):
Summary:
Hurst et al. developed a new Tol2-based transgenesis system ImPaqT, an Immunological toolkit for PaqCl-based Golden Gate Assembly of Tol2 Transgenes, to facilitate the production of transgenic zebrafish lines. This Golden Gate assembly-based approach relies on only a short 4-base pair overhang sequence in their final construct, and the insertion construct and backbone vector can be assembled in a single-tube reaction using PaqCl and ligase. This approach can also be expandable by introducing new overhang sequences while maintaining compatibility with existing ImPaqT constructs, allowing users to add fragments as needed.
Strengths:
The generation of several lines of transgenic zebrafish for the immunologic study demonstrates the feasibility of the ImPaqT in vivo. The lineage tracing of macrophages by …
Reviewer #2 (Public review):
Summary:
Hurst et al. developed a new Tol2-based transgenesis system ImPaqT, an Immunological toolkit for PaqCl-based Golden Gate Assembly of Tol2 Transgenes, to facilitate the production of transgenic zebrafish lines. This Golden Gate assembly-based approach relies on only a short 4-base pair overhang sequence in their final construct, and the insertion construct and backbone vector can be assembled in a single-tube reaction using PaqCl and ligase. This approach can also be expandable by introducing new overhang sequences while maintaining compatibility with existing ImPaqT constructs, allowing users to add fragments as needed.
Strengths:
The generation of several lines of transgenic zebrafish for the immunologic study demonstrates the feasibility of the ImPaqT in vivo. The lineage tracing of macrophages by LPS injection shows this approach's functionality, validating its usage in vivo.
Weaknesses:
(1) There is no quantitative data analysis showing the percentage of off-target based on these 4-bp overhang sequences.
(2) There is no statement for the upper limitation of the expandability.
(3) There is no data about any potential side effect on their endogenous function of promoter/protein of interest with the ImPaqT method.
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