Rapid generation of single‐insertion transgenics by Tol2 transposition in zebrafish.

Background: The Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of the transgenic cassette in F1 fish. Each of these transgenes is susceptible to positional effects from the surrounding regulatory landscape, which can lead to altered expression and, consequently, activity. Scientists therefore must strike a balance between the need to maximize reproducibility by establishing single‐insertion transgenic lines and the need to complete experiments within a reasonable timeframe. Results: In this article, we introduce a simple competitive dilution strategy for rapid generation of single‐insertion transgenics. By using cry:BFP reporter plasmid as a competitor, we achieved a nearly fourfold reduction in the number of the transgene of interest integrations while simultaneously increasing the proportion of single‐insertion F1 generation transgenics to over 50%. We also observed variations in transgene of interest expression among independent single‐insertion transgenics, highlighting that the commonly used ubiquitous ubb promoter is susceptible to position effects. Conclusions: Wide application of our competitive dilution strategy will save time, reduce animal usage, and improve reproducibility of zebrafish research. Key Findings: Competitive dilution of Tol2 transgenesis vectors facilitates isolation of single‐insertion transgenic zebrafish lines in the F1 generation, reducing animal usage, improving reproducibility, and saving time. [ABSTRACT FROM AUTHOR]