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Efficient knock-in method enabling lineage tracing in zebrafish.

Jiarui Mi1, Olov Andersson2

  • 1Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

Life Science Alliance
|March 6, 2023
PubMed
Summary

We developed a new, efficient cloning-free method for zebrafish gene knock-in using DNA donors. This technique enables precise genetic labeling and lineage tracing, revealing new insights into pancreatic and liver cell development.

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Area of Science:

  • Developmental Biology
  • Genetics
  • Zebrafish Models

Background:

  • Precise genetic modification is crucial for understanding gene function and developmental processes.
  • Existing zebrafish knock-in methods can be inefficient or disrupt endogenous gene expression.

Purpose of the Study:

  • To develop a cloning-free 3' knock-in strategy for zebrafish that is efficient and avoids disrupting targeted genes.
  • To create reporter lines for endogenous gene expression and enable lineage tracing studies.

Main Methods:

  • Utilized PCR-amplified double-stranded DNA (dsDNA) donors with genetic cassettes for fluorescent proteins and Cre recombinase.
  • Employed 5' end-protected primers for enhanced PCR amplicon integration efficiency.
  • Coinjected dsDNA donors with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration.

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  • Targeted four genetic loci (krt92, nkx6.1, krt4, id2a) to generate knock-in lines.

    • Main Results:

    • Successfully generated 10 knock-in zebrafish lines serving as reporters for endogenous gene expression.
    • Lineage tracing using iCre or CreERT2 knock-in lines revealed that nkx6.1+ cells are multipotent pancreatic progenitors.
    • Identified id2a+ cells as multipotent in both liver and pancreas, restricting to ductal cells.
    • Demonstrated progenitor properties in hepatic id2a+ ducts following severe hepatocyte loss.

    Conclusions:

    • The developed cloning-free 3' knock-in strategy is efficient and straightforward for zebrafish.
    • This technique facilitates cellular labeling and lineage tracing, providing valuable insights into developmental cell dynamics.
    • The generated reporter lines offer new tools for studying pancreatic and liver development and regeneration.