title: Functional screen of zebrafish deubiquitylating enzymes by morpholino knockdown and in situ hybridization.
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.
authors: Craniofacial Developmental Biology Laboratory, Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114, USA. kftse@aori.u-tokyo.ac.jp
authors: William Ka Fai
authors: WK
authors: Tse
authors: Yun-Jin
authors: YJ
authors: Jiang
mesh_terms: D000818:Animals; D001483:Base Sequence; D003890:Desiccation; D004625:Embryo, Nonmammalian; D010450:Endopeptidases / Q000235:genetics*; D055785:Gene Knockdown Techniques; D017403:In Situ Hybridization / Q000379:methods; D008845:Microinjections; D060172:Morpholinos / Q000008:administration & dosage / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D060172:Morpholinos; D029961:Zebrafish Proteins; D010450:Endopeptidases