The Tg.AC mouse model is being evaluated by the National Toxicology Program (NTP) as an adjunct to the conventional two-year bioassay. The short term 26-week bioassay relies on the empirical observation that Tg.AC transgenic mice produce skin papillomas when topically treated with chemical carcinogens. Created on the FVB/N mouse, only one of five founder lines displayed the unique characteristics now associated with the Tg.AC mouse. The laboratory's research goal is to understand the induced tumorigenic response of Tg.AC mice and the dependence on the v-Ha-ras transgene. The transgene, was thought be ectopically expressed because of the chromosomal integration site. We have found this not to be the case. The unique expression pattern of the transgene in Tg.AC mice is dependant upon a palindromic arrangement of two transgenes arranged head to head within the integration site. A small loss of DNA sequence, due to deletions in the promoter juncture region, abrogate gene expression. We have developed an assay to detect these deletion events and implemented this assay into a genotypic screen of Tg.AC breeders. To investigate the role of the palindromic region in the expression of the transgene we have characterized the mutant frequency of naturally occurring mutants in Tg.AC x FVB/N out crosses. The mutant frequency was determined to be 1.5+/- 2%. Using Tar Cloning we have cloned the integration site and transgene, and reintroduced the clones into FVB/N carcinoma cell lines. Transgene expression was evident in all cells regardless of flanking DNA content, implicating a region within the transgene integration site and consistent with the role of the palindromic region. DNase I foot printing and gel shift experiments indicate multiple transcription factor binding sites concentrated near the axis of symmetry of the palindrome as well as the basal promoter region. These factors are also present in leukemia cells, which also express the transgene but are not present in normal non-transgene expressing skin. Finally, we have completed our first gene array experiments to investigate gene expression changes prior to and following transgene activation. This work indicates that few gene expression changes are evident between Tg.AC and FVB/N mice when hair follicle cycle differences are eliminated.
