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CMV Promoter-Driven Expression and Visualization of Tagged Proteins in Live and Fixed Zebrafish Embryonic Epidermis

  • Kirti Gupta
  • Mahendra Sonawane
Protocol
  • 29 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

In the recent past, zebrafish has emerged as a highly useful vertebrate model for biomedical research. Owing to its easy handling, suitability for high-throughput genetic and chemical screens, and tractability by high-resolution microscopy, it is possible to study molecular mechanisms of vertebrate development and disease biology using zebrafish. This chapter introduces zebrafish epidermis as a model for studying cell biology of epithelial tissues in vivo and describes the technique of protein expression using plasmid vectors having cytomegalovirus (CMV) promoter. It details protocols for microinjection of plasmids into fertilized zebrafish oocytes, screening of the epidermal clones expressing the tagged protein, immunostaining, and mounting of embryos for both fixed and live imaging by confocal microscopy. This collection of protocols allows for analysis of localization of a wide range of proteins including those involved in intracellular transport, cell polarity, cell adhesion, and so on, during early developmental stages of zebrafish embryos and larvae.

Keywords

Zebrafish Microinjections CMV promoter–based expression Live imaging Confocal microscopy 

Notes

Acknowledgments

We would like to thank Sumit Sen for the illustrations, Dr. Clyde Pinto for contributing to Fig. 5, Geetika Chouhan and Mandar Phatak for critically reading the manuscript, and Kalidas Kohale for fish maintenance. This work was supported by funding from TIFR-DAE (12P-121).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Kirti Gupta
    • 1
  • Mahendra Sonawane
    • 1
  1. 1.Department of Biological SciencesTata Institute of Fundamental ResearchMumbaiIndia

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