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Functional Screen of Zebrafish Deubiquitylating Enzymes by Morpholino Knockdown and In Situ Hybridization

  • William Ka Fai Tse
  • Yun-Jin Jiang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 815)

Abstract

In order to unfold the function of genes, solely performing mRNA over-expression is not enough nowadays. Traditional protein expression experiments, such as Western blotting and immunohistochemical staining, could only provide researchers the changes of expression levels and/or location of their targets. To make a more strong and convincing statement about gene function, it is necessary to perform both “gain-of-function” and “loss-of-function” studies. Both assays can be performed easily by transfecting DNA plasmid and siRNA in cell culture system; while in zebrafish, mRNA and morpholino (MO) microinjection can serve similar purposes. It is common for the zebrafish community to carry out microinjection experiments to explore a gene function. Instead of making a single knockdown/over-expression of a gene, we foresee that more and more large-scale screens on certain protein families will be performed in the future. Here, based on our previous experience in zebrafish “loss-of-function” screening on deubiquitylating enzymes, we describe a general work flow, from morpholino designation, in situ hybridization, to data analysis, as a reference for researchers who may be interested in a similar screen.

Key words

Deubiquitylating enzymes In situ hybridization “Loss-of-function” screening Morpholino Zebrafish 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Craniofacial Developmental Biology Laboratory,Center for Regenerative Medicine, Massachusetts General HospitalHarvard Medical SchoolBostonUSA
  2. 2.Institute of Molecular and Genomic Medicine, National Health Research InstitutesMiaoli CountyTaiwan

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