PUBLICATION
Functional screen of zebrafish deubiquitylating enzymes by morpholino knockdown and in situ hybridization
- Authors
- Tse, W.K., and Jiang, Y.J.
- ID
- ZDB-PUB-111205-7
- 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.
Citation
Tse, W.K., and Jiang, Y.J. (2012) Functional screen of zebrafish deubiquitylating enzymes by morpholino knockdown and in situ hybridization. Methods in molecular biology (Clifton, N.J.). 815:321-331.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping