Regulation Of Transgene Expression

我们实验室的研究目标是更好地了解化学品或环境损伤引发的肿瘤发生过程中的分子机制。为了实现这一目标，我们开发了一种称为Tg. AC的转基因小鼠品系。将Tg.AC小鼠改造为含有v-Ha-ras癌基因，已发现其功能与人类大多数上皮肿瘤相关或致病。转基因小鼠在受伤、局部用化学致癌物处理或暴露于紫外线辐射时产生皮肤乳头状瘤。当用非致癌物质治疗时，它们不会产生肿瘤。由于这种独特的表型，国家毒理学计划（NTP）正在评价Tg.AC小鼠模型，作为鉴别致癌物的常规两年生物测定的辅助方法。为了帮助进行该评价，我们开发了对Tg.AC小鼠的育种者进行基因分型的试验，以确保反应性。此外，我们已经从分子水平上描述了转基因在致瘤反应中的作用。为了了解基因表达的调控，我们绘制了启动子和潜在的转录因子及其结合位点。我们已经克隆并测序了小鼠11号染色体上的整合位点。我们实验室最近的数据表明，转基因整合在一个新的基因。我们已经鉴定了该基因，并正在研究其在转基因调控中的表达和调控作用。我们还利用ras依赖的肿瘤发生反应的Tg.AC小鼠的遗传方法，试图了解肿瘤发生的重要信号通路。我们发现，小鼠缺乏KSR（Ras激酶抑制因子），这是EGFr介导的信号传导所需的基因产物。当KSR缺陷型小鼠与Tg.AC小鼠杂交时，TPA依赖性致瘤反应被阻断。提示EGFr/ras/KSR信号通路在Tg.AC肿瘤发生中起重要作用。最后，我们继续使用基因阵列技术来鉴定在ras介导的肿瘤发生中起重要作用的新基因。

Our laboratory's research goal is to gain greater understanding of the molecular mechanisms that function during the tumorigenic process initiated by chemicals or environmental insults. To accomplish this goal we have exploited a transgenic mouse line called Tg.AC. Tg.AC mice were engineered to contain a v-Ha-ras oncogene, whose function has been found to be associated or causal of a majority of epithelial tumors in humans. Tg.AC transgenic mice produce skin papillomas when wounded, topically treated with chemical carcinogens, or exposed to ultraviolet radiation. They do not produce tumors when treated with non-carcinogens. As a result of this unique phenotype, the Tg.AC mouse model is being evaluated by the National Toxicology Program (NTP) as an adjunct to the conventional two-year bioassay for the identification of carcinogens. To aid in this evaluation we have developed assays to genotype the breeders of Tg.AC mice to ensure responsiveness. In addition, we have molecularly characterized the transgene's role in the tumorigenic response. To understand the regulation of gene expression we have mapped the promoter and potential transcription factors and their binding sites. We have cloned and sequenced the integration site on mouse chromosome 11. Recent data from our laboratory indicate that the transgene is integrated in a novel gene. We have identified this gene and are investigating the expression and regulatory role it may have in regulating of the transgene. We are also exploiting the ras dependent tumorigenic response of Tg.AC mice in genetic approaches in an attempt to understand the signaling pathways important for tumorigenesis. We have found that mice deficient in KSR (Kinase Suppressor of Ras) , a gene product required for EGFr mediated signaling. When KSR deficient mice are crossed to Tg.AC mice the TPA dependant tumorigenic response is blocked. This data indicate that the EGFr/ras/KSR signaling pathway is very important in Tg.AC tumorigenesis. Lastly, we continue to use the gene array technology to identify new genes that play significant roles ras mediated tumorigenesis.