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Cancer Research , 20 , Evans H.

Mutation Research , 31 , Scott D. J, Danford N. Part 1 revised. Freshney R. Basic Principles of Cell Culture. In Culture of Cells for Tissue Engineering. Vunjak-Novakovic G. Corby N Hone P. Int J Radiat. Goto K. Chromosoma Berl. Marshall R. Mutagenesis 18 , — Edwards A. Radiation Protection Dosimetry , — An International System for Cytogenetic Nomenclature Hardnen D. Karger A. Richardson C. Part Hilliard C. Greenwood S. Environ and Mol. Mutagenesis 31 , — Doak S.

Cancer Res. Ishidate M. Environ Mol Mutagenesis 10 , Suppl 10, 1— In: Sheila M. While pronuclear injection is usually impractical, as fish nuclei are difficult to locate, there are exceptions e. Where the chorion poses a significant obstacle to microinjection, it may be removed mechanically or chemically, its hardening may be prevented, the DNA may be injected through the micropile, or a pilot hole may be made by microsurgery.

The zebrafish Brachydanio rerio has been used to produce stable lines that exhibit reproducible patterns of transgene expression. See Stuart, Gary W..

The In Vitro Mammalian Chromosome Aberration Test

They are much less expensive to buy and raise than any mammalian species. They are extremely fecund, oviparous and are externally fertilized. Because of these factors, it is much less expensive and technically less complicated to perform gene transfer procedures on them. Their eggs are transparent and embryonic development occurs at a much faster rate than in the mouse. Large scale production of homozygous diploid zebrafish can be obtained in a reproducible and relatively simple manner. See Streisinger, G. The fact that fish are aquatic organisms cannot be overlooked. This has important implications with respect to simplifying both experimental design and implementation.

Fish present clear advantages. Certain enzymes are known to play a role in the conversion of promutagens into mutagens. If a particular species of animal, such as a mouse, is deficient in a particular enzyme of this type, it may be modified, by crossbreeding or genetic engineering, to provide. For example, a transgenic animal may be produced that features a P enzyme missing in the mouse, or homologous recombination may be used to replace , it with a human counterpart' or to insert a stronger promoter upstream of a gene encoding such an enzyme.


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A further consideration is the spontaneous mutation rate in the host animal. Preferably, this background level of mutation is low, e. The natural environment of an animal may make it better suited for testing certain scenarios of chemical exposure. For example, waterborne chemical are preferably tested using transgenic fish or amphibia or aquatic mammals. If an animal is particularly sensitive to mutagens, it may be useful in detecting less potent mutagens. A final issue is the economic importance of the animal. A chemical which has a detrimental effect on an economically important animal may be rejected even if it does not have a serious adverse effect on humans.

This could be the case with, for example, honey bees, or with fish. The laboratory mouse has been the most popular host animal for use in the development of transgenic animals, as there are numerous strains available. Mice are, of course, the most widely available laboratory animal, and many strains are available. However, there are no substantial restrictions on the use of other laboratory or livestock species in such work.


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Among the higher mammals, pigs are preferred, and fish offer an interesting alternative to mammalian subjects. While the DNA may plainly contain bacterial genes, procaryotic vector DNA more particularly any prokaryotic replicon should be removed before the transgene is introduced into the host cell s to be developed into a transgenic animal.

For example, the inclusion of large flanking sequences of lambda DNA in early beta-globin transgene constructs apparently inhibited expression of the transgene in transgenic mice. See Wagner, et al.

The most common technique for the production of transgenic animals involves the microinjection of the transgene into the pronucleus of fertilized eggs. These mice usually exhibit the same degree of mosaicism in somatic and germ cells, but in some mice the germ cells may totally lack the transgene.

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In the latter case, the mice will be unable to transmit the transgene to their progeny. One of the requirements for successful pronuclear microinjection is the ability to locate the pronucleus. Eggs of some species are more opaque than others. The employment of art-recognized techniques of facilitating microinjection is within the invention as contemplated. Transgenes may also be incorporated into the host cell genome by microinjection of DNA into the cytoplasm of fertilized or unfertilized eggs, into the nuclei of two-cell embryos, or into the blastocoel cavity.

Mosaicism is more prevalent with these approaches. Alternatives to microinjection include electroporation, liposome-mediated entry, and particle gun bombardment. Preimplantation embryos may also be infected with retroviruses engineered to carry the transgene. This method has found particular favor for the production of transgenic birds. Still another method for the production of transgenic animals is to introduce the transgene, on-a suitable vector, into totipotent teratocarcinoma or embryonic stem cells and then incorporate these cells into embryos. Once transgenic animals are produced, they or their transgenic progeny are exposed to the suspect chemical.

The exposure may be by ingestion, inhalation, injection, or skin contact. The dosage employed may be one comparable to that experienced by the target species in the environment of interest, or it may be a higher dose, in order to provide a margin of safety. After an appropriate exposure period, the animals are examined to determine whether the marker gene has been mutated. In one embodiment, the marker gene confers a phenotype which can be detected without killing the animal, e.

For this to work, the transgene or its expression product must have a characterizing feature which is recognizable by a detectably labeled homing agent. For example, monoclonal antibodies may be prepared which bind a wild-type polypeptide, in preference to the mutant polypeptide encoded by the marker gene. These antibodies may be detectably labeled and injected into the animal. If the epitopes for these antibodies are reestablished by specific reverse mutation, by mutation, these antibodies may be localized by scintigraphic means known in the art. A forward assay can also be envisioned, but is less desirable because of the increased background.

Certain cells may, of course, be removed without killing the transgenic animal. These include blood cells, skin cells, mucosal cells, etc. Such cells may be removed and examined as described below. However, this method, while permitting the monitoring of the development of the mutagenic effect of the chemical in certain tissues over time, does not provide information as to mutagenesis of the marker gene in all tissues and organs.

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Therefore, in another and preferred embodiment, the animal is sacrificed so that all of its tissues and organs of interest may be examined for mutation of the transgene. In this embodiment, it is not strictly necessary that the animal have expressed the marker gene. However, it is preferable that the animal express the marker gene, since mutation rates may be different for expressed and unexpressed genes. Mellon, et al. In an especially preferred embodiment, transgenic mice which are homozygous for lad are mated with transgenic mice which are homozygous for lacZ under lacO control.

The progeny are hemizygous for a single copy of lad and for one or two copies of lacZ. The progeny animals are exposed to the potential mutagen. If the lad gene is mutated, the cells of the progeny animal will stain blue since lacZ gene is then derepressed. Having more than one copy of the lad gene is undesirable, since then both copies must be mutated in order to derepress the lacZ gene. To avoid the problem of a mutation in lacZ that prevents its expression hiding the status of the lad marker gene, it is preferable to use two copies of the lacZ gene.

However, more than two copies is undesirable as this would titrate out the repressor. A variety of other phenotypic characteristics could be used to identify cells containing a mutagenized form of the marker gene. These include antibody sensitivity or resistance, antigenicity, etc. See discussion of marker genes above. Alternatively, the marker gene may be recovered from the genomic DNA of the transgenic animal.