Assessing metabolic rates in zebrafish using a 3D-printed intermittent-flow respirometer and swim tunnel system.

Zebrafish have become a widely used vertebrate model in physiology and reliable measures of their metabolic rate are needed. We have developed a 3D-printed respirometer and swim tunnel system and used it for obtaining accurate measurement of standard metabolic rate (SMR) and maximal, aerobic metabolic rate (MMR) in zebrafish under rest and maximal exercise, respectively. We compared a slow (stepwise) protocol to a fast (continuous) protocol for determining MMR.

Generation and validation of a myoglobin knockout zebrafish model.

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.

Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension.

We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.