In the last 10 years, the zebrafish (Danio rerio) has emerged as an important genetic model for studying vertebrate development and neurobiology. This fish are small, develop rapidly (for a vertebrate), and produce many offspring. They have transparent embryos that make it easy to observe abnormalities in early development. In a typical large-scale forward screen for recessive mutations in the zebra fish, a parental generation male is exposed to tank water containing a chemical mutagen (ENU, ethylnitrosourea), which produces mainly base substitutions.
Each of the male’s sperm potentially carries a different set of base substitution mutations. Each F1 contains one normal genome (from the mother) and one mutagenized genome (from the father). F1 males are crossed individually to wild females and descendants allowed to interbreed randomly in the same tank. The inbreeding allows any recessive mutation to come to homozygosity.
Some examples of phenotypic mutants that arise in such screens are shown in Figure 16-12. The above screen can be lengthy and laborious; however, special tricks can speed up the identification of mutations in zebra fish. One of these is the creation of haploid fish. To create haploid fish, males are treated with large doses of UV light. The exposed sperm nuclei become so heavily mutagenized that they are unable to contribute their genome to the zygote. However, they are still able to penetrate the egg membrane and activate the development of the haploid oocyte nucleus. The haploid fish that are produced typically fail to form adults, but they do survive for several days, and this immature fish can be assayed for recessive phenotypes (Figure 16-13).
The geneticist is thus able to focus on those tanks pro- during interesting mutations, and the mutants can be recovered by inbreeding the parental F1 female. Another way to speed up the screen is to use a molecular tag. Earlier we saw that transposons can be used as tags. In zebra fish, retrovirus vectors serve as tags. Recall that retroviruses are RNA viruses that use reverse transcriptase to create a double-stranded DNA copy of their viral genome. The virus is injected into embryos of size 1000–2000 cells. When retroviral DNA is replicated in a cell, the new DNA becomes integrated at random into the host chromosomes.
The retroviral DNA causes mutations by gene disruption and also serves as a convenient molecular tag for the disrupted genes. The resulting fish are subjected to an inbreeding program as before to bring mutations to homozygosity. Once a mutant is identified in a subsequent generation, the gene that it has inserted into can be determined from its sequence, determined with the help of PCR. The viral insert serves as a primer that allows amplification of the sequence into the adjacent region.
In higher eukaryotes, many DNA regulatory sequences act as enhancers to control transcription (as we saw in Chapter 10). In model organisms such as Drosophila, screens can be designed to hunt for these enhancers. These regulatory elements enhance transcription of any gene whose transcription start site is nearby, so the strategy is to randomly insert a transgenic reporter construct designed to respond to any nearby enhancer. The construct will have a transcription start site and a “reporter” gene such as green fluorescent protein (GFP) or blue dye–producing galactosidase.
The construct is carried on a transposon. Crosses are made that mobilize this re- porter construct so that it transposes itself into various sites in the genome, and then the distribution of the re- porter protein product is observed. By this means, we can identify the locations of enhancer elements that drive a particular pattern of gene expression (Figure 16-14). The reporter-transgene insertions are called enhancer traps. Suppose that one particular reporter insertion is expressed only in developing Drosophila eye tissue.
We can infer that it is likely that a gene expressed in the eye resides in the vicinity. Thus, the neighbouring genes are candidates for involvement in some aspect of eye development and can be isolated and studied.
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