title: Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.
abstract: The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: F O
authors.initials: FO
authors.lastname: Kok
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: M
authors.initials: M
authors.lastname: Shin
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: C-W
authors.initials: CW
authors.lastname: Ni
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: A
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Grosse
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3584CT Utrecht, Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: van Impel
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3584CT Utrecht, Netherlands.
authors.forename: B C
authors.initials: BC
authors.lastname: Kirchmaier
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3584CT Utrecht, Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Peterson-Maduro
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: G
authors.initials: G
authors.lastname: Kourkoulis
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: I
authors.initials: I
authors.lastname: Male
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: D F
authors.initials: DF
authors.lastname: DeSantis
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: S
authors.initials: S
authors.lastname: Sheppard-Tindell
authors.affiliation: Uppsala University, Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, 751 85 Uppsala, Sweden.|Karolinska Institute, Department of Medicinal Biochemistry and Biophysics, 171 77, Stockholm, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Ebarasi
authors.affiliation: Uppsala University, Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, 751 85 Uppsala, Sweden.|Karolinska Institute, Department of Medicinal Biochemistry and Biophysics, 171 77, Stockholm, Sweden.
authors.forename: C
authors.initials: C
authors.lastname: Betsholtz
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3584CT Utrecht, Netherlands.|Institute for Cardiovascular Organogenesis and Regeneration, Cells-in-motion Cluster of Excellence, University of Münster, 48149 Germany.
authors.forename: S
authors.initials: S
authors.lastname: Schulte-Merker
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Wolfe
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: N D
authors.initials: ND
authors.lastname: Lawson
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques / Q000379:methods*; D060172:Morpholinos / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*
chemical_list: D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D029961:Zebrafish Proteins; D003851:Deoxyribonucleases