This type of screen saves one generation by screening F1 fish directly and reduces the number of fish required to survey a given number of haploid genomes. Haploid development is initiated by inactivating sperm by UV irradiation and using this sperm to fertilize eggs in vitro. Haploid embryos develop essentially as normal for the first few days, but have a few consistent defects, including short tails, deformed ear capsules, and edema, by early larval stages, after which they die. Therefore, a haploid-based screen for mutations in organogenesis and late differentiating structures would be inadvisable. However, mutations that alter the basic structure of the zebrafish body plan are easily identifiable, and most of the mutations previously identified in zebrafish have been identified by haploid screening of X-ray-induced mutations (14-16).
The ease with which haploids may be produced depends entirely on the ability of mutagenized F1 females to yield their stored eggs, a trait that seems highly strain-dependent. Typically, in a strain considered accessible to this manipulation, sexually mature gravid females will yield eggs in at least 20-30% of squeeze attempts. Each wild-type strain should be assessed for its ability to undergo this procedure. Ideally, fish of the AB strain should be used, since they have been selected over several generations for the ability to allow in vitro fertilization in this manner. Tricks that seem to lead to a more consistent yield from females are:
1. The separation of males from females the night before squeezing to prevent the males from inducing spawning in the females.
2. Squeezing females early in the morning, since they are induced to spawn when the lights come on. Since sperm has to be collected and inactivated prior to females being squeezed, placing female fish in an enclosed light cycle that comes on later than the main facility may help this procedure.
Production of haploid embryos requires little or no specialized equipment (4). A shortwave UV source and anesthetic are all that is required. Sperm may be collected from males and used to fertilize eggs in vitro in the following way:
1. Anesthetize sexually mature males by placing them in a 0.002% (w/v) tricaine solution until gill movement has stopped. Rinse anesthetized fish in water.
2. Place ventral side up in a moist sponge bed designed to keep the fish wedged upright. The genital opening is located between the pelvic fins and should be wiped dry before sperm collection.
3. With Millipore forceps, gently squeeze this area while collecting sperm in a glass capillary pipet attached by thin-gage tubing to a mouth pipet or other suction device.
4. Place sperm in a solution of full-strength Hank's on ice, and store until enough has been collected in the vial to turn the solution opaque and slightly milky. Approximately 0.5 mL are needed for 20 fertilizations.
5. Transfer the Hank's/sperm solution on a watch glass or small plastic Petri dish, and UV-irradiate. Inactivated sperm should be stored on ice. Since individual sources vary in their output of UV irradiation, a time-course of irradiation should be performed. However, as a reference point, we find that irradiation for 5 min at a distance of 20 cm by our UV shortwave source produces inactivated sperm still able to fertilize. Shorter periods fail to inactivate and lead to diploid embryos, easily discernable by their longer tails. Longer periods highly reduced fertility.
6. Anesthetize females, and then dry initially by flipping the fish on a clean paper towel.
7. Place in a clean, small plastic Petri dish. Apply even pressure to the belly, pushing toward the genital opening. Hold the back of the fish gently with the other hand. Undue pressure will lead to internal hemorrhaging and death, so a desire to yield eggs must be tempered against the need to keep the fish alive, no matter how gravid a female may appear. Increased pressure will never lead to an increased percentage of fish that yield eggs. Also, the success of a haploid-based screen relies on the female surviving, since she carries the only recoverable chromosomes in the screen. This risk can be circumvented by separating expressed eggs into dishes, fertilizing one with wild-type, unirradiated sperm, and keeping these out-crossed embryos until the results of a screen are known.
8. Females often yield eggs that cannot be fertilized. These eggs appear milky and opaque when they are squeezed into the Petri dish. Fertile eggs appear shiny yellow and complete in appearance. Separate eggs from the female using a clean spatula, and add 50-100 pL of the Hank's/sperm solution, followed immediately with 0.5 mL of water. Eggs must not be left to dry and should be covered. Sperm solution should be added as quickly as possible after the eggs have been expressed.
9. After 1 min add 3-4 mL of water, and swirl the fertilized eggs gently to avoid clumping.
10. Monitor eggs to see if the chorions raise. Separate from infertiles, and allow to develop.
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