PUBLICATION

Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish

Authors
Burg, L., Palmer, N., Kikhi, K., Miroshnik, E.S., Rueckert, H., Gaddy, E., MacPherson Cunningham, C., Mattonet, K., Lai, S.L., Marín-Juez, R., Waring, R.B., Stainier, D.Y.R., Balciunas, D.
ID
ZDB-PUB-181115-11
Date
2018
Source
PLoS Genetics   14: e1007754 (Journal)
Registered Authors
Balciunas, Darius, Lai, Shih-Lei (Ben), Marín-Juez, Rubén, Stainier, Didier
Keywords
none
MeSH Terms
  • Alleles
  • Animals
  • Base Sequence
  • DNA Transposable Elements
  • Genome
  • Homologous Recombination*
  • Introns
  • Mutagenesis*
  • Mutation
  • Oligonucleotides*/genetics
  • Reproducibility of Results
  • T-Box Domain Proteins/genetics
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics
(all 14)
PubMed
30427827 Full text @ PLoS Genet.
Abstract
Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.
Genes / Markers
Marker Marker Type Name
aldh1a2GENEaldehyde dehydrogenase 1 family, member A2
ift70GENEintraflagellar transport 70
tbx18GENET-box transcription factor 18
tbx20GENET-box transcription factor 20
tcf21GENEtranscription factor 21
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Figures
Figure Gallery (3 images)
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Expression
Phenotype
Fish Conditions Stage Phenotype Figure
aldh1a2tpl137/+; aldh1a2tpl138/+standard conditionsPrim-15
aldh1a2tpl137/+; aldh1a2tpl138/+standard conditionsProtruding-mouth
aldh1a2tpl137/+; aldh1a2tpl138/+standard conditionsProtruding-mouth
aldh1a2tpl137/+; aldh1a2tpl138/+standard conditionsProtruding-mouth
aldh1a2tpl137/+; aldh1a2tpl138/+standard conditionsProtruding-mouth
tbx20tpl122/+; tbx20tpl145Tg/+; zf2148Tg/+chemical treatment by environment: tamoxifenLong-pec
tbx20tpl122/+; tbx20tpl145Tg/+; zf2149Tg/+chemical treatment by environment: tamoxifenLong-pec
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Mutations / Transgenics
Allele Construct Type Affected Genomic Region
tpl19GtTransgenic Insertion
tpl19RGtTransgenic Insertion
tpl119
    		Unknown
    tpl122
      		Small Deletion
      tpl135TgTransgenic Insertion
      tpl136TgTransgenic Insertion
      tpl137
        		Small Deletion
        tpl138
          		Small Deletion
          tpl139TgTransgenic Insertion
          tpl140TgTransgenic Insertion
          1 - 10 of 15
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          Human Disease / Model
          No data available
          Sequence Targeting Reagents
          Target Reagent Reagent Type
          aldh1a2CRISPR11-aldh1a2CRISPR
          aldh1a2CRISPR12-aldh1a2CRISPR
          aldh1a2CRISPR9-aldh1a2CRISPR
          aldh1a2CRISPR10-aldh1a2CRISPR
          ift70CRISPR1-ift70CRISPR
          tbx20CRISPR2-tbx20CRISPR
          tbx20CRISPR3-tbx20CRISPR
          tcf21CRISPR2-tcf21CRISPR
          tcf21CRISPR3-tcf21CRISPR
          1 - 9 of 9
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          Fish
          Fish
          aldh1a2tpl137/+; aldh1a2tpl138/+
          tbx20tpl122/+; tbx20tpl145Tg/+; zf2148Tg/+
          tbx20tpl122/+; tbx20tpl145Tg/+; zf2149Tg/+
          WT
          1 - 4 of 4
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          Antibodies
          No data available
          Orthology
          No data available
          Engineered Foreign Genes
          Marker Marker Type Name
          CreEFGCre
          EGFPEFGEGFP
          GAL4EFGGAL4
          1 - 3 of 3
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          Mapping
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          Citation
          Burg, L., Palmer, N., Kikhi, K., Miroshnik, E.S., Rueckert, H., Gaddy, E., MacPherson Cunningham, C., Mattonet, K., Lai, S.L., Marín-Juez, R., Waring, R.B., Stainier, D.Y.R., Balciunas, D. (2018) Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish. PLoS Genetics. 14:e1007754.