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
- PubMed
- 30427827 Full text @ PLoS Genet.
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.
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
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping