Notes

1. An in-depth guide to zebrafish raising and techniques has been provided in The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio), University of Oregon Press (12). This book provides an invaluable reference for most experimental manipulations in zebrafish.

2. Raising of zebrafish embryos has traditionally been done in a defined salt solution or embryo medium. We find that this is not necessary as long as an antifungal agent, such as methylene blue, is added to system's water. This may of course vary with the quality of individual water sources.

3. It may be advantageous for some researchers to screen for mutations in pigmentless embryos. In wild-type embryos, pigment develops during the early pharyngula period of development. By early hatching, pigment can significantly reduce the information gained from optical inspection of the embryo. Although a number of fully viable pigment mutations exist in the appropriate genetic background, the researcher has the option of using PTU to inhibit pigment synthesis (see Subheading 2.). This may be of use when rescreening mutations to determine effects later in development.

4. It is important to realize that many different regulations govern the importation of fish into different countries, and obtaining import licences can often be costly and time-consuming. It is important to investigate these procedures to avoid fish being impounded and delays that could lead to death of transported fish.

5. Many regulations also govern the mutagenesis of vertebrate animals. In many instances mutagenesis of zebrafish requires a licence from the appropriate authority. Mutagenesis without this licence may be illegal.

6. X- or y-ray mutagenesis and PCR-based screening: A number of studies have indicated the efficacy of X- and y-rays in inducing mutations within the germline of zebrafish, although at a significantly lower efficiency than the above-described treatment with ENU (8). X- and y-rays induce mutation by producing doublestrand breaks within the DNA, and often result in deletion or rearrangement of large regions of the chromosome. This complexity may reduce overall viability of F1 embryos or affect the segregation of mutations in the adult germline. This may explain the lower frequency of recovered mutations. Inducing translocations and deletions, however, is desirable in screens that utilize PCR to detect gene-specific lesions or as a reference point for cloning the gene represented by a given mutation. Mutations can be easily induced in adult fish or in collected sperm, and a brief method for mutagenesis is described below. It is important to use a calibrated X- or y-ray source with a wide beam to ensure whole-sample irradiation.

a. Sperm can be collected as outlined above, irradiated while being kept on ice and resuspended in 10% Hank's solution in small glass vials. Alternatively, adult male fish, pretested for high fertility, can be anesthetized in tricane and placed on a moist sponge bed to constrain the fish within the path of the beam.

b. The ability of a given source to induce mutations should ideally be calibrated, but 200-300 rads seem to be sufficient to induce mutations.

c. Sperm after irradiation can be added directly to eggs obtained from squeezed females, or irradiated fish can be treated similarly to those treated with ENU to produce F1 founder fish.

It is theoretically possible to search for specific DNA lesions generated by X- or y-rays via the use of PCR. Primers that span a region of interest can be used to amplify target sequences from haploid embryos derived from F1 founder fish generated from X or y-rays mutagenized fish or sperm. Although the chances of locating a rearrangement that breaks specifically in a known gene are small, it is possible to identify deletions that span a gene region seemingly with fairly high frequency. This is dependent on the dose of radiation that is used to induce such rearrangements, and it is quite likely that complex rearrangements are induced by high doses of X- or y-rays. Such complex rearrangements invariably led to complex phenotypes, and it is difficult to say with certainty that a given pheno-type results from rearrangement in a specific gene region. It is thus yet to be determined if PCR can be used to identify gene-specific lesions efficiently by this method. It can be used to check quickly if an identified phenotype results from the deletion of a gene of interest, and it may be in this application that PCR-based screening is most useful, rather than its systematic application to every brood of a random screen.

The ability to generate a number of gene-specific PCR markers in any one PCR reaction allows multiple genes to be screened at any one time for a given phenotype. The screen procedure first involves the preparation of DNA from fish demonstrating a given phenotype. PCR is performed on this DNA and scored for the presence or absence of a DNA marker. PCR can also be used to map quickly the limits of a deletion and assess its suitability for further genetic analysis, such as the saturation mutagenesis of a specific region.

7. Stock maintenance: The protocols required for general fish raising and maintenance have been listed in detail elsewhere (12), but it is worthwhile to mention a few tips that specifically impinge on the efficiency of a small-scale mutagenic screen. Mutations will be identified in an ENU screen on average once per one to two haploid genomes screened (8,9). There is a great temptation to keep every mutation that is identified from such a screen. However, the maintenance of these mutations as stocks can quickly cut into available tank space in a small facility. It is far more efficient to make a decision about mutations when they are identified. Alternatively, mutations can be stored as frozen sperm and can be recovered by in vitro fertilization of squeezed wild-type eggs with the thawed sperm. Care should be taken to assay the protocol for the ability of thawed sperm to fertilize by initially utilizing wild-type sperm in test fertilizations.

Freezing sperm:

a. Sperm is collected using the methods outlined above. Sperm from at least four males identified as heterozygotes for the mutation of interest should be used.

b. Sperm is added to 4-5 vol of fish Ringers containing 10% methanol and 15% powdered nonfat milk.

c. The solution is frozen in capillary tubes placed in 10-mL plastic centrifuge tubes (Sorvall) by incubation in dry ice for 20 min.

d. Capillary tubes are stored under liquid nitrogen after freezing.

8. Morphological screening: Marker-based screening is most efficient when coupled with a morphological screen. Zebrafish embryos are amenable to screening at most stages of development. If a researcher is interested in processes during organogenesis at stages later than 2 d it may be advisable to perform mutagenesis in a strain mutant for pigment synthesis, such as golden or albino, since this greatly increases the ability to detect subtle defects in organogenesis. The rapid development of the zebrafish embryo can often make screening at some stages inconvenient. The ability to screen some stages may rely on placing the fish "off cycle" by utilizing time-regulated light sources for tanks so that fish are induced to lay at specific times. Researchers should make a checklist of structures and processes that are of interest and screen accordingly. After early pharyngula stages embryos should be anesthetized in tricaine to stop twitching and movement to best reveal structure. A subset of embryos should be dechorinated, but the dorsal aspects of the embryo are best revealed by rolling the embryo within the chorion to present the dorsal surface. Later stages require anesthetizing also, since by 3 d fry are able to swim rapidly and can be frustrating to screen.

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