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
References
Dec 22, 1998·Molecular and Cellular Biology·Y KamachiH Kondoh
May 12, 2000·Biochimica Et Biophysica Acta·J Summerton
Oct 4, 2000·Nature Genetics·A Nasevicius, S C Ekker
Jul 31, 2001·Genesis : the Journal of Genetics and Development·S C Ekker, J D Larson
Oct 28, 2003·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Karen A UrtishakSteven A Farber
Jul 9, 2004·Briefings in Functional Genomics & Proteomics·Saulius Sumanas, Jon D Larson
Oct 12, 2004·BMC Developmental Biology·Karen K Nelson, Richard W Nelson
Dec 18, 2004·Methods in Cell Biology·Stephen C Ekker
Jan 13, 2006·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Yuich OkudaYusuke Kamachi
Jan 18, 2006·BMC Developmental Biology·Vinzenz LinkCarl-Philipp Heisenberg
Apr 11, 2006·Developmental Biology·Zac PujicJarema Malicki
Nov 14, 2006·Biochemical and Biophysical Research Communications·Shu TsuruzoeMitsuyoshi Nakao
Apr 14, 2007·Current Topics in Medicinal Chemistry·James E Summerton
Citations
May 16, 2013·Molecular Biotechnology·Noriko UmemotoToshio Tanaka
Sep 26, 2013·Chemical Communications : Chem Comm·Fazel Shabanpoor, Michael J Gait
Apr 16, 2010·Zebrafish·Ilya A Shestopalov, James K Chen
Oct 29, 2008·BMC Biotechnology·Francisca Alcaraz-PérezMaría L Cayuela
Jan 27, 2012·Development·Anna TrofkaScott A Holley
May 14, 2010·PLoS Genetics·Yuichi OkudaYusuke Kamachi
Jun 6, 2009·PloS One·Emil KaraulanovChristof Niehrs
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
Jan 28, 2010·Plant Biotechnology Journal·Robert G BirchAndreas S Betzner
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
Feb 20, 2019·ELife·Rodrigo M YoungStephen W Wilson
Jun 5, 2017·Experimental Eye Research·Ryne A GorsuchDavid R Hyde
Oct 28, 2021·Journal of the American Chemical Society·Kristie DarrahAlexander Deiters
Feb 20, 2016·Genome Research·Nicolas ChassaingPatrick Calvas
Jul 28, 2021·Annual Review of Genetics·Gabrielius Jakutis, Didier Y R Stainier