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ZFIN ID: ZDB-PUB-111205-7
Functional screen of zebrafish deubiquitylating enzymes by morpholino knockdown and in situ hybridization
Tse, W.K., and Jiang, Y.J.
Date: 2012
Source: Methods in molecular biology (Clifton, N.J.)   815: 321-331 (Chapter)
Registered Authors: Jiang, Yun-Jin, Tse, Ka Fai William
Keywords: none
MeSH Terms:
  • Animals
  • Base Sequence
  • Desiccation
  • Embryo, Nonmammalian
  • Endopeptidases/genetics*
  • Gene Knockdown Techniques*
  • In Situ Hybridization/methods
  • Microinjections
  • Morpholinos/administration & dosage
  • Morpholinos/genetics
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics*
PubMed: 22131002 Full text @ Meth. Mol. Biol.
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.