The genotoxic impact of environmental chemicals upon human health and well- being is well appreciated. Numerous short-term bacterial and mammalian cell mutagenesis tests have contributed to our understanding of the mutagenicity of many agents. However, the inability to incorporate tissue differences in P450 activities into their design has been a major shortcoming. The ability to produce transgenic mice with reporter genes that can monitor mutagenic activity in tissues and organs of tester animals represents a significant technological advance. However, the existing animal models also have drawbacks. These include 1) the use of multiple transgene copies per cell; 2) the use of bacterial genes that are subject ot methylation; 3) the use of genes not expressed in the mammalian host; 4) the need to recover these transgenes from the mammalian genome and express them in E. coli; 5) the inability to identify mutant cell types within an organ; 6) the inability to identify some major classes of mutational activity including recombination, chromosome loss, large deletions. The four research projects in this program are complementary in their respective approaches and all address the above-mentioned concerns in one capacity or another. Project 1) Dr. Stringer will use a small transgene with attendant introns and exons encoding human placental alkaline phosphatase (PAP), which expresses ubiquitously and whose activity can be detected histochemically. Mice carrying mutant genes that require a transition, a frameshift or a recombinational event to reestablish enzyme activity have been or will be produced. This strategy provides a basis for in situ detection of these mutagenic events, either spontaneous or mutagen- induced. Project 2) To circumvent the need for extrinsic transgenes, Dr. Stambrook will modify an endogenous gene at its normal chromosomal position as a reporter of mutagenic activity. The gene is that which encodes adenine phosphoribosyltrans-ferase (APRT), a purine salvage enzyme that concerts adenine to AMP. Dr. Stambrook will use gene targeting in ES cells to introduce inactivating point mutations into Aprt. Cells undergoing reverse mutation will regain APRT function and will acquire the capacity to sequester tagged adenine ina the form of labeled AMP which will be incorporated into nucleic acids and mark the cell. Project 3) A major mutagenic event not detected by existing rodent models is loss of heterozygosity (LOH). Dr. Tischfield will use existing mice, heterozygous at Aprt, to define the frequency of LOH in different tissues at a neutral locus and to determine whether mutagens and carcinogens may alter this frequency. Project 4) Dr. Doetschman will provide mice with a TGF-beta1 null background and cross mutant PAP transgene and mutant Aprt into this background and others (eg. p53) to determine the effect of genetic background on LOH and mutation frequency in situ. He will also produce a p27 knock out mouse into which the reporters will be crossed.
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