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Generation and validation of a myoglobin knockout zebrafish model
Rasmus Hejlesen1,2, Kasper Kjær-Sørensen2, Angela Fago3
1Department of Biology, Zoophysiology, Aarhus University, Aarhus, Denmark.
Abstract:
Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.
Insights
Researchers generated three new zebrafish myoglobin (Mb) knockout models. The mb(Auzf13.2) line showed no developmental or adult phenotypes, suggesting it is a reliable tool for studying Mb's biological role.
Area of Science:
- Genetics and Molecular Biology
- Developmental Biology
- Zebrafish Models
Background:
- Conflicting results exist regarding in vivo phenotypes of myoglobin (Mb) loss.
- Previous studies in knockout mice and knockdown zebrafish yielded inconsistent data.
- A well-characterized Mb knockout model is needed to clarify its biological role.
Purpose of the Study:
- To generate and characterize novel zebrafish myoglobin (mb) knockout lines.
- To assess the in vivo phenotypes associated with Mb loss during development and in adulthood.
- To validate a specific knockout line for future biological studies.
Main Methods:
- Utilized the CRISPR/Cas system to generate three distinct zebrafish mb knockout lines.
- Evaluated morphological phenotypes, developmental length, and lethality in embryonic and larval stages.
- Assessed adult homozygous knockout fish for Mb protein absence, mRNA degradation, viability, length, heart size, and gene expression via transcriptomic analysis.
- Screened for off-target mutations in 36 loci.
Main Results:
- None of the three generated mb knockout lines exhibited morphological phenotypes, developmental changes, or lethality.
- The adult mb(Auzf13.2) homozygous knockout line lacked Mb protein and showed near-complete mb mRNA degradation.
- Adult mb(Auzf13.2) knockout fish displayed no changes in viability, length, or heart size.
- Transcriptomic analysis revealed no altered gene expression in adult heart tissue of mb knockout fish.
- No off-target mutations were detected in the 36 screened loci.
Conclusions:
- Three distinct zebrafish mb knockout lines were successfully generated with no observable phenotypes during development.
- The mb(Auzf13.2) line was validated as a robust model, showing no signs of genetic compensation or off-target effects in adult hearts.
- The mb(Auzf13.2) zebrafish line is a promising candidate for future investigations into the biological functions of myoglobin.
