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A Simple and Efficient Microinjection Protocol for Making Transgenic Sticklebacks

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Gasterosteus aculeatus, the threespine stickleback, has been used for many years to study vertebrate behavior, physiology, evolution and ecology. Further studies of this organism at the molecular level would be greatly enhanced by methods to increase or decrease the activity of specific genes, or to transfer genes between fish with different morphologies. We have tested a variety of methods for microinjection of DNA into 1-2 cell stickleback embryos. Holding the embryos in place using a custom-made glass chamber made it possible to penetrate the chorion with a glass capillary needle, and inject small volumes of DNA solutions into the blastodisc region of the fertilized egg. To test for expression and maintenance of DNA following injection, we injected a DNA plasmid containing the zebrafish muscle specific (α) actin promoter fused to the coding region of a jellyfish green fluorescent protein (GFP) gene, together with the rare cutting endonuclease, ISceI. Injected sticklebacks expressed the GFP reporter gene in developing muscle, maintained expression for at least 6 months, and transmitted the GFP construct to their own progeny when raised to sexual maturity. Further use of this method should make it possible to introduce a variety of constructs into developing sticklebacks and to study the molecular basis of the interesting morphological, behavioral, and physiological differences among recently evolved stickleback forms.

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