PUBLICATION

Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay

Authors
Kamachi, Y., Okuda, Y., and Kondoh, H.
ID
ZDB-PUB-080218-3
Date
2008
Source
Genesis (New York, N.Y. : 2000)   46(1): 1-7 (Journal)
Registered Authors
Kamachi, Yusuke, Kondoh, Hisato
Keywords
morpholino antisense oligonucleotides, sox2, chordin, knockdown efficiency, mis-targeting
MeSH Terms
  • Animals
  • Base Sequence
  • Embryonic Development
  • Gene Expression Regulation, Developmental
  • Genetic Techniques*
  • Luciferases/metabolism*
  • Models, Genetic
  • Molecular Sequence Data
  • Oligonucleotides, Antisense/chemistry*
  • Phenotype
  • Zebrafish/embryology*
  • Zebrafish/genetics*
PubMed
18196596 Full text @ Genesis
Abstract
Despite the broad use of morpholino antisense oligonucleotides (MO) to knockdown gene function in zebrafish embryos, the efficiency of this method has not been successfully assessed, particularly in the cases of translation-blocking MOs. In our current study, we describe a luciferase assay-based system that can monitor the MO knockdown levels in zebrafish by the use of a fusion reporter construct containing the 5'-mRNA sequence of the gene of interest and the luciferase coding sequence. The decrease in luciferase activity in zebrafish embryos that have been coinjected with this reporter RNA construct and a MO that targets the gene of interest correlated well with the level of inhibition of the corresponding endogenous protein synthesis, and also with the appearance of a knockdown phenotype. This indicates the usefulness of our method. We have also found that MOs can exert considerable knockdown effects upon unintended gene targets if 15 bases or longer of contiguous homology exists between these genes and the 25-base MO in question. Our quantitative assessment method also reveals, however, that an effective and specific knockdown can be achieved when employing a strategy that takes advantage of the synergistic effect of double MOs used at low levels.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes