Differentially Expressed Genes in the Skin of Tumor Susceptible K14-Agouti Transgenic Mice . Mus musculus

The mouse agouti gene is normally expressed in the skin and regulates coat color pigmentation. Dominant regulatory mutations in the agouti gene, such as viable-yellow (Avy), cause ubiquitous over-expression of the wild-type gene product in every tissue in the body. As a result of this altered agouti expression, viable-yellow mice have solid yellow hair, they are obese and diabetic, and they have increased susceptibility to spontaneous and chemically induced tumors in a wide variety of tissues (e.g., skin, lung, liver, mammary gland and urinary bladder). Additionally, liver-specific expression of the agouti gene was shown to promote hepatocellular carcinogenesis, even in the absence of obesity and diabetes. Our lab recently extended these findings to show that the agouti gene also promotes skin cancer in the absence of obesity and diabetes. In K14-Agouti transgenic mice, the wild-type agouti gene is over-expressed in the skin under the regulatory control of the keratin 14 (K14) promoter. Over-expression of agouti in the skin results in yellow coat color without the obesity and diabetes. In two-stage skin carcinogenesis experiments the agouti protein was shown to act as a tumor promoter since it promotes the development of skin tumors in the K14-Agouti transgenic mice, but only after the skin was initiated with a single dose of 7, 12-dimethylbenz[a]anthrance (DMBA). These experiments set the stage for determining the mechanism of action of agouti protein in tumor promotion, which remains completely uncharacterized. As a first step in determining the role of agouti in skin cancer, we used cDNA microarray and quantitative real time polymerase chain reaction (QRT-PCR) analysis to identify and validate a set of differentially expressed genes in the skin of K14-Agouti transgenic mice at the promotion stage of carcinogenesis. Keywords: promotion stage of carcinogenesis, biological replicates, Overall design: Total RNA from three different eight-week-old male K14-Agouti transgenic mice was hybridized individually against a pooled total RNA sample from three sex-matched, age-matched non-agouti control mice. All three hybridizations were repeated with dye swapping to minimize dye bias in the microarray analysis. Since each one of the NIA 15K mouse ESTs is spotted in duplicate on the microarray slides, 12 data points were generated for the expression level of each gene