The objectives of my project were two-fold. The first was to develop techniques to generate zebrafish embryos that transiently express apoaequorin, the protein part of the Ca²⁺-senstive bioluminescent complex, aequorin, and to make use of these techniques to optimize protocols for introducing the luminophore co-factor, coelenterazine, into zebrafish embryos, so as to reconstitute active aequorin in vivo for the purpose of in situ Ca²⁺ imaging. The second part of my project was to apply the in vivo aequorin reconstitution techniques developed during the first part of my project, to reconstitute aequorin in stable germ-line apoaequorin transgenic zebrafish....[ Read more ]

The objectives of my project were two-fold. The first was to develop techniques to generate zebrafish embryos that transiently express apoaequorin, the protein part of the Ca²⁺-senstive bioluminescent complex, aequorin, and to make use of these techniques to optimize protocols for introducing the luminophore co-factor, coelenterazine, into zebrafish embryos, so as to reconstitute active aequorin in vivo for the purpose of in situ Ca²⁺ imaging. The second part of my project was to apply the in vivo aequorin reconstitution techniques developed during the first part of my project, to reconstitute aequorin in stable germ-line apoaequorin transgenic zebrafish.

During the course of my project, I developed: (1) in vitro aequorin reconstitution assay protocols for estimating levels of in vivo apoaequorin expression, (2) coelenterazine incubation protocols for early stage embryos and (3) a pericardial-injection technique for loading coelenterazine into late stage embryos.

The results from the first part of my project clearly indicated that it was possible to express and reconstitute aequorin in vivo using mRNA-based transient expression techniques, and that by applying this methodology, the period of aequorin-based Ca²⁺ imaging could be extended in the developing zebrafish to 48 hpf. This is an additional 24 hours compared to what can be achieved by injecting embryos with aequorin at the single-cell stage.

The objectives of the second part of my project were somewhat compromised when it was discovered that a non-functional apoaequorin was being expressed in our stable transgenic germ-line fish. Investigation indicated that this was due to founder fish being transfected with a truncated DNA plasmid, missing key portions of the apoaequorin DNA nucleotide sequence. In spite of this, several key experimental protocols developed during the transient expression phase of my project were applied successfully to the stable germ-line fish. This work will thus serve as a foundation for the eventual generation of apoaequorin expressing stable germ-line fish.