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

Genesis : the Journal of Genetics and Development
Yusuke KamachiHisato Kondoh

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 ...Continue Reading

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Citations

May 16, 2013·Molecular Biotechnology·Noriko UmemotoToshio Tanaka
Apr 16, 2010·Zebrafish·Ilya A Shestopalov, James K Chen
Oct 29, 2008·BMC Biotechnology·Francisca Alcaraz-PérezMaría L Cayuela
Aug 26, 2014·Neuroscience Research·Kazuhiro OgaiSatoru Kato
Feb 5, 2009·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Eri OguraYusuke Kamachi
Jul 18, 2008·Current Protocols in Molecular Biology·Jon D Moulton, Yi-Lin Yan
Nov 12, 2015·Frontiers in Immunology·Hetron Mweemba Munang'anduØystein Evensen
Oct 28, 2021·Journal of the American Chemical Society·Kristie DarrahAlexander Deiters
Jul 28, 2021·Annual Review of Genetics·Gabrielius Jakutis, Didier Y R Stainier

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