PUBLICATION

Use of target protector morpholinos to analyze the physiological roles of specific miRNA-mRNA pairs in vivo

Authors
Staton, A.A., and Giraldez, A.J.
ID
ZDB-PUB-111205-14
Date
2011
Source
Nature Protocols   6(12): 2035-2049 (Journal)
Registered Authors
Giraldez, Antonio, Staton, Alison
Keywords
genetic analysis, nucleic acid based molecular biology
MeSH Terms
  • 3' Untranslated Regions
  • Animals
  • Binding, Competitive
  • Embryo, Nonmammalian
  • Gene Expression Regulation
  • Genetic Techniques
  • Luciferases/analysis
  • MicroRNAs/chemistry
  • MicroRNAs/physiology*
  • Morpholinos/chemistry*
  • RNA, Messenger/chemistry
  • RNA, Messenger/physiology*
  • Zebrafish/embryology
  • Zebrafish/genetics*
PubMed
22134127 Full text @ Nat. Protoc.
Abstract
MicroRNAs (miRNAs) regulate gene expression by pairing with complementary sequences in the 32 untranslated regions (UTRs) of transcripts. Although the molecular mechanism underlying miRNA biogenesis and activity is becoming better understood, determining the physiological role of the interaction of an miRNA with its target remains a challenge. A number of methods have been developed to inhibit individual miRNAs, but it can be difficult to determine which specific targets are responsible for any observed phenotypes. To address this problem, we use target protector (TP) morpholinos that interfere with a single miRNA-mRNA pair by binding specifically to the miRNA target sequence in the 32 UTR. In this protocol, we detail the steps for identifying miRNA targets, validating their regulation and using TPs to interrogate their function in zebrafish. Depending on the biological context, this set of experiments can be completed in 6–8 weeks
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping