Premium
Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay
Author(s) -
Kamachi Yusuke,
Okuda Yuichi,
Kondoh Hisato
Publication year - 2008
Publication title -
genesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.093
H-Index - 110
eISSN - 1526-968X
pISSN - 1526-954X
DOI - 10.1002/dvg.20361
Subject(s) - gene knockdown , morpholino , luciferase , zebrafish , oligonucleotide , biology , reporter gene , gene , microbiology and biotechnology , gene expression , genetics , transfection
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. genesis 46:1–7, 2008. © 2008 Wiley‐Liss, Inc.