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Related Experiment Video

Updated: Mar 20, 2026

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks
08:51

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Published on: May 13, 2016

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Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks.

Priscilla A Erickson1, Nicholas A Ellis1, Craig T Miller2

  • 1Department of Molecular and Cell Biology, University of California, Berkeley.

Journal of Visualized Experiments : Jove
|May 24, 2016
PubMed
Summary

This study details a microinjection protocol for threespine stickleback embryos, enabling genetic studies of evolved traits. This method facilitates gene editing and transgenesis for understanding adaptation in this model organism.

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Area of Science:

  • Evolutionary biology
  • Genetics
  • Developmental biology

Background:

  • Threespine stickleback fish offer a unique vertebrate model for studying the genetic basis of diverse evolved phenotypes.
  • Adaptation to various freshwater environments has resulted in remarkable phenotypic diversity in marine stickleback populations.
  • The ability to cross marine and freshwater forms, coupled with available genomic resources, facilitates genetic mapping of evolved traits.

Purpose of the Study:

  • To present a detailed protocol for microinjecting nucleic acids into threespine stickleback embryos.
  • To enable functional genetic studies, including transgenesis and genome editing, for investigating gene function and regulation.
  • To facilitate the study of gene expression and function in the context of evolutionary adaptation.

Main Methods:

  • Microinjection of nucleic acids (DNA, RNA, CRISPR/Cas9 reagents) into fertilized one-cell stickleback embryos.
  • Utilizing the Tol2 transposase system for integrating transgenes (reporter plasmids, BACs) into the genome.
  • Employing TALEN and CRISPR/Cas9 genome editing tools to induce targeted mutations in candidate genes and cis-regulatory elements.
  • Developing techniques to assess transgenesis success and recover stable transgenic lines.

Main Results:

  • A robust protocol for microinjection into challenging stickleback embryos (thick chorion, small blastomeres) is described.
  • The protocol supports the generation of transgenic reporter lines for studying gene regulation.
  • The protocol enables targeted gene editing to functionally assess candidate genes and regulatory elements involved in adaptation.
  • Methods for assessing transgenesis and establishing stable lines are provided.

Conclusions:

  • The described microinjection protocol overcomes technical challenges, making threespine stickleback a more accessible model for genetic research.
  • This methodology empowers researchers to perform functional genetic analyses, advancing our understanding of the genetic underpinnings of adaptation.
  • The protocol facilitates the study of gene function and regulation, crucial for dissecting the evolution of complex phenotypes in sticklebacks.