Authors:

Carolina Kim

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About Paper:

Calcium ions play an essential role in cellular and physiological functions such as stimulus-response reactions and homeostasis by serving as a second messenger in many signaling pathways. Researchers have been using genetically encoded calcium indicators (GECIs) to monitor their activities in vivo to facilitate studying the functions of calcium. Along this direction, GCaMP series GECIs have been extensively developed and successfully applied to many cell types, especially neurons. Recently a new group of calcium sensors, NEMO, were reported to have a superior dynamic range compared to the most recently developed GCaMP8. However, more research and trials are still needed to obtain a holistic view of NEMO's full potential in monitoring calcium activity in different model organisms. In this study, we aim to create a NEMOs transgenic zebrafish line by Tol2 transgenesis. First, we codon-optimized NEMOs for zebrafish, synthesized an open reading frame, and cloned it into the Gateway middle entry vector. Then, we created a final plasmid construct with Tol2 minimum sequences by LR gateway recombination. Lastly, we microinjected this final Tol2 construct with NEMOs into the 1-cell stage zebrafish embryos. Green fluorescent signals were able to be detected. These results suggested that we have created a functional zebrafish codon-optimized NEMOs, which could be used for generating a stable transgenic zebrafish line. In the future, NEMOs fish lines can be compared to GCaMP indicators and be used for exploring other embryonic and physiological questions, such as adult fin regeneration.

Source:

Purdue University / 2023

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Co-authors:

Carolina Kim