1. Fish strains (see Notes 3 and 4): Strains are available from laboratories on request. The choice of strain to mutagenize is extremely important, since particular strains have been used for mapping and some are more amenable to genetic manipulations. The inbred strain recommended for mutagenesis is one derived originally by the late George Streisinger (University of Oregon) and is designated AB, although other laboratories have developed their own inbred strains (8,9). The advantages of AB over other strains are to be found primarily in haploid screening. AB fish have been selected over many generations for their ability to provide eggs for fertilization in vitro and for the lack of embryonic lethal mutations within the genetic background. Further, the most extensive genetic map for zebrafish, using polymorphic variation in the PCR products generated by random primers, has used AB as its inbred strain (11). In the absence of information about the amount of polymorphic variation between other strains and AB, it would seem useful to induce mutations in this strain to aid in mapping.

2. Mutagens: The choice of mutagen will determine the type and number of mutants you find. ENU (Sigma, St. Louis, MO) has proven to be the most efficient mutagen for the induction of mutations within the zebrafish germline (8,9). It is highly mutagenic and carcinogenic, and must be handled with extreme care. Whenever possible, dedicated sets of equipment and tanks must be used, and all mutagenic procedures carried out in a suitable fume hood. All solutions and equipment contaminated with ENU are inactivated by incubation in a 10% solution of sodium thiosulfate, adjusted to pH 10.0 with sodium hydroxide, for at least 24 h at room temperature. To create the mutagenic solution, ENU is dissolved directly in the ampule in 10 mM acetic acid to a concentration of 100 mM and stored at -20°C as a stock solution (ENU activity is highly pH-dependent; it is higher with increasing pH and a correspondingly higher lethality). Immediately prior to use, the stock solution is thawed and diluted to 3-10 mM sodium phosphate buffer (pH 6.6) to make the final mutagenic ENU solution (see Notes 5 and 6).

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