Generation and validation of a myoglobin knockout zebrafish model.

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.