A robust knock-in approach using a minimal promoter and a minicircle.

Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.

A robust knock-in approach using a minimal promoter and a minicircle.

Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.

Voltage-gated sodium channel scn8a is required for innervation and regeneration of amputated adult zebrafish fins.

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.