Enhancing the Efficiency ofN-Ethyl-N-Nitrosourea–Induced Mutagenesis in the Zebrafish

Abstract

N-Ethyl-N-nitrosourea (ENU) treatment is the standard experimental method used for chemical mutagenesis of many vertebrate organisms commonly used in the laboratory. In zebrafish and medaka, the use of a repetitive, sublethal dose of 3-3.5 mM ENU has been shown to give the best balance between mutagenicity and toxicity. However, even at this concentration, a significant proportion of fish die during the treatment. Therefore, large numbers of fish are required to obtain a sufficient number of mutagenized founders at the end of the procedure. Additionally, it is quite common to have high levels of mortality in any particular dosing cycle. This may cause a mutagenesis experiment to suddenly fail after several weeks of work. Here we provide a very simple method for ENU mutagenesis of zebrafish using a subparalytic dose of clove oil as a sedative that drastically reduces the lethality of ENU treatment in fish. This facilitates ENU mutagenesis protocols considerably, facilitates higher dosing, and allows for sensitive strains of fish such as homozygous mutants to be mutagenized for use in genetic studies.

How does mutation affect the distribution of phenotypes?

“…Individual males and females of the WIK (Wild India Kolkata) strain of Danio rerio (Rauch et al 1997;Trevarrow and Robison 2004) were randomly paired and bred. Repeated doses of ENU induce more mutations than a single dose at a higher concentration (Hitotsumachi et al 1985;Rohner et al 2011), suggesting that mutation rate is lowest at the first treatment, potentially due to more effective mutation repair mechanisms after a single dose (Noveroske et al 2000). In forward genetic screens, which aim to induce mutations in all genes, zebrafish are exposed to 3 mM ENU for one hour each week for six consecutive weeks, inducing approximately one mutation every 1.0 × 10 5 -1.5 × 10 5 base pairs (de Bruijn et al 2009;Rohner et al 2011).…”

“…Each of these full-sibling families contributed sons to both a control and a mutagenesis treatment, where males were exposed to ENU. In forward genetic screens, which aim to induce mutations in all genes, zebrafish are exposed to 3 mM ENU for one hour each week for six consecutive weeks, inducing approximately one mutation every 1.0 × 10 5 -1.5 × 10 5 base pairs (de Bruijn et al 2009;Rohner et al 2011). Here, our goal was to induce fewer mutations, more similar to long-term mutation accumulation experiments; we therefore exposed fish to 3 mM ENU for one hour in a one-off treatment.…”

“…The actual number of mutations was likely to have been substantially less than this. Repeated doses of ENU induce more mutations than a single dose at a higher concentration (Hitotsumachi et al 1985;Rohner et al 2011), suggesting that mutation rate is lowest at the first treatment, potentially due to more effective mutation repair mechanisms after a single dose (Noveroske et al 2000).…”

“…The single dose ENU treatment was applied as described in Rohner et al (2011). To ensure only germline mutations were transmitted, we allowed at least two weeks between ENU treatment and breeding (Mullins et al 1994;Solnica-Krezel et al 1994).…”

Celsr1a is essential for tissue homeostasis and onset of aging phenotypes in the zebrafish

“…Tuebingen male fish were treated with N-ethyl-N-nitrosourea (ENU; Sigma) following an optimized 5 protocol using clove oil (Sigma) as a sedative (Rohner et al, 2011). The surviving mutagenized 6 founders were crossed to frnt t31786 homozygous females.…”