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Heritable Targeted Inactivation of the Rainbow Trout (Oncorhynchus mykiss) Master Sex-Determining Gene Using Zinc-Finger Nucleases
- Source :
- Marine Biotechnology, Marine Biotechnology, Springer Verlag, 2014, 16 (2), pp.243-250. ⟨10.1007/s10126-013-9546-8⟩
- Publication Year :
- 2013
- Publisher :
- Springer Science and Business Media LLC, 2013.
-
Abstract
- Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.
- Subjects :
- Male
endocrine system
Molecular Sequence Data
Biology
medicine.disease_cause
Applied Microbiology and Biotechnology
gene knockout
Frameshift mutation
03 medical and health sciences
Transforming Growth Factor beta
medicine
Animals
sdY
14. Life underwater
Genes, sry
Gene
Gene knockout
030304 developmental biology
Genetics
Sex Characteristics
0303 health sciences
Mutation
Base Sequence
[SDV.BA]Life Sciences [q-bio]/Animal biology
Gene targeting
Zinc Fingers
[SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology
04 agricultural and veterinary sciences
Sex Determination Processes
Zebrafish Proteins
Sex reversal
Endonucleases
rainbow trout
Stop codon
Oncorhynchus mykiss
Gene Targeting
040102 fisheries
0401 agriculture, forestry, and fisheries
Female
Rainbow trout
Genetic Engineering
zfn
Subjects
Details
- ISSN :
- 14362236 and 14362228
- Volume :
- 16
- Database :
- OpenAIRE
- Journal :
- Marine Biotechnology
- Accession number :
- edsair.doi.dedup.....d14e220f67f629fb56b4c12c85b5b0f1