Recessive Lethal Mutations - Essay Example

Recessive Lethal Mutations George Streisinger pioneered the potential of zebrafish as a vertebrate organism suitable for forward genetics screening research, about twenty years ago. In the laboratory environment zebrafish are easily handled. The combination of embryological and genetic zebrafish leads to external development of transparent embryo, which allows fundermental vertebrate development process from gastrulartion to organogenesis to be visualized and studied. Also the heartbeats and the blood circulation of the embryoare readily observed. Mutation can be induced with high frequency in zebrafish and recessive mutation can be recovered within two generations. Although, there is a primary method for identifying the genetic basis of phenotypes in invertebrate organism, while genetic screening for vertebrate organism is rarely perform. This is because the number of animal to be raise, maintained and screened are very large for a moderate-size screen and million to achieve saturation. Zebrafish and mice have been highly successful and two large screening have been carried out in zebrafish. The simple visual screening of embryo in the first 5 days after fertilization in zebrafish, can reveal mutation in genes essential for normal development of most of the major organ system such as heart, blood, gut liver jaws, eyes, and ears. Genetic basis of many additional phenotypes important in vertebrate development, physiology and behavior are revealed on future screening. In order to understand how genes specify a biological process of both phenotypes that can occure and the number of genes involve, it is important identifying the mutated genes. Because high mutation frequencies can be obtain with chemical mutagens. Actually the mutation frequency can vary widely for different loci, chemical mutagens can induce mutation in most genes. With all these advantages, there is still some disadvantage associate with it because cloning mutant gene is difficult, as these chemicals usually cause point mutations. Positioning cloning chemical induce mutants are made feasible over time, but cloning remain expensive and laborious in vertebrate animal with large genomes. A less effective approach to chemical mutagenesis that greatly speeds the cloning of mutant genes is known as insertional mutagenesis. The integration of exogenous DNA sequences into a genome can be mutagenic, and the inserted DNA serves as a tag to clone mutated genes, though fewer lesions are obtained per genome and also insertional mutagens seldom, if integrated randomly into host DNA. When large-scale genetic screening is carried out, it results in successful identification of many genes that define embryological pathways. However, two scientists from Boston and Tubingen are inspired by the remarkable characteristic of the zebrafish, along with the first zebrafish genetic screening identified mutant embryonic phenotype in F3 generation. Some of the mutated developmental genes identified in the two screens have been cloned, which assist in the dissection of the gene network that controls the early development. For example, the mutated genes in the endoderm mutants such as casanova, bonnie and clyde, and faust can be assembled into a genetic pathway that have been shown tom encode transcription factors that are necessary for endoderm formation. Analysis of proviral insertions has revealed that different germ cell are infected independently in F1 progeny and with high titer virus stocks they often have multiple integrations and any given insertion is transmitted mosaically to between 1% and 40% of the F1 pronegy. Individual F1 fish can inherit multiple insertion and proviral insertions in F1 fish and transmitted in a mendelian fashion. When outcrossing founder fish, identified F1 fish with single proviral insertion, generated an F2 family for each insertion, and then inbred transgenic F2 fish and examined F3 progeny to identify mutation. This system is not too efficient for large-scale screen because each insertion was inbred individually, therefore require it own F2 family and a separate tank F1 fish are enrich with multiple insertion and use these to generate F2 families where many insertion can be screened simultaneously. The diagram above give the insight design for the protocol devise for large, diploid insertion mutagenesis screens. The rates at which fish reach sexual maturity depend on the size of the screen, which is base on the number of F1 and F2 families our facility could accommodate. It is highly necessary to have excellent founder fish and method for selecting F1 fish with the maximum number of unique insert. The component of a large scale screening protocol of zebrafish in clude: 1. The preparation of high titer storcks of virus, using two viruses to generate the founder fish. By preparing a cell line producing high titer virus to obtain a packaging cell line, infect it with a virus, and select a clone of cell line that yield virus with high titer on both mouse and fish cell line. 2. Monitoring the successful injection of virus by the embryo assay inother yo assess the efficiency with which injected embryos are infected, we used either quantitative Southern blotting or quantitative PCR. After every injection, several injected embryo were lysed and there DNA are extracted for analysis every two to five days. Two genomic sequences were probed, a single-locus gene RAG2 and proviral sequences. The ratio of these signals was normalized to signals from DNA of a fish heterozygous for a single insertion. The result of the embryo assay value was use as a measure of the average number of proviral integrations per cell. Injected eggs that were raised were assumed to have the same embryo assay value as those that were sampled from the same injected batch. Founder fish from batches of injected embryos with a range of embryo assay values were tested to determine the amount of provirus they could transmit to their F1. We outcrossed the founders and used the quantitative assay for RAG2 versus proviral sequences on DNA extracted from pools of their F1 progeny. Actually there was considerable variation between founders from injections that had yielded the same embryo assay value, it show a definite correlation between embryo assay and average provirus transmission rate. 3. Mating founder fish with each other carries out F1 family generation; there is considerable variation in the number of inserts between fish in a single F1 family, as well as between F1 families. Inorder to identify fish with the greatest number of inserts in each family in the Southern blot analysis. The generation of F1 fish shows that the greatest numbers of inserts are often derived from the same germ cell(s) and hence share proviral insertions. 4. To generating F2 families, multi-insert F1 fish are mated and 50-70 embryos from each pair are raised by performing sibling crosses of F2 fish at three months age or older. To determine that insertions segregating in an F2 family is linked to an identified mutation, Southern analysis is performed on DNA extracted from fin clips of parents of all the crosses screened in the family. Both parent of every cross that showed the phenotype have specific insert (Southern band) that must be shared and must be in only one or neither of the parents of all crosses that did not show the phenotype. Screening F3 embryos, and demonstrating that mutants are caused by proviral insertions. From these screening protocols it is observed that the prototype of the recessive embryonic lather mutant obtained in the large screen to date are shown in the figure below as found in the chemical mutagenesis screens where many embryonic lethal mutations are relatively nonspecific. These may be as result of mutations in genes, which are widely expressed in the embryo and required for cell survival or growth. Some mutants display highly specific defects. For example, nearly normal mutant embryos appear normal at 5 days of age, except that in the majority of homozygotes, the swim bladder fails to inflate and the embryos die. The result of a specific phenotype result from a mutation in a wide expressed houskeeping type gene EF1. The bleached blond mutant embryos have striking specific defects in the appearance of pigmentation in their melanocytes and in the pigmented epithelium in the retina, apparently due to a mutation in the gene encoding the Ac45 subunit of the vacuolar ATP synthase. There are other defects as well, however, because the majority of embryos fail to develop a swim bladder and those that do develop fail to thrive, although they can survive for many days. hi43 mutant embryos are generally normal, for example, head and jaw structures appear well developed, unlike most pleiotropic mutants; however, the liver is clearly abnormal, appearing dark and lacking circulation, and the gut appears compressed, although it is unclear whether or not this is a consequence of the degree of unconsumed yolk. References 1. M.. Allende, A., Amsterdam, T. Becker, K. Kawakami, N. Gaiano, and N. Hopkins. 1996. Insertional mutagenesis in zebrafish identifies two novel genes. 2. Bai, C., P. Sen, K. Hofmann, L. Ma, M. Goebl, J.W. Harper, and S.J. Elledge. 1996. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86: 263-274 3. Baier, H., S. Klosterman, T. Trowe, R.O. Karlstrom, C. Nusslein-Volhard, and F. Bonhoeffer. 1996. Genetic dissection of the retinotectal projection. Development 123: 415-425 4. Gaiano, N., A. Amsterdam, K. Kawakami, M. Allende, T. Becker, and N. Hopkins. 1996b. Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature 383: 829-832 5. Gossler, A., A.L. Joyner, J. Rossant, and W.C. Skarnes. 1989. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244: 463-465 6. Grunwald, D.J. and G. Streisinger. 1992. Induction of recessive lethal specific locus mutations in the zebrafish with ethylnitrosourea. Genet. Res. 59: 103-116 7. Miyoshi, H., M. Takahashi, F.H. Gage, and I.M. Verma. 1997. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc. Natl. Acad. Sci. 94: 10319-10323 Read More