Regulation of Transgene Expression

国家毒理学计划（NTP）正在评价Tg.AC小鼠模型，作为常规两年生物测定的辅助方法。 短期26周生物测定依赖于经验观察，即Tg.AC转基因小鼠在局部用化学致癌物处理时产生皮肤乳头状瘤。在FVB/N小鼠上创建的五个创始品系中只有一个显示出现在与Tg.AC小鼠相关的独特特征。本实验室的研究目标是了解Tg.AC小鼠诱导的致瘤反应和对v-Ha-ras转基因的依赖性。 由于染色体整合位点的存在，转基因被认为是异位表达。 我们发现情况并非如此。转基因在Tg.AC小鼠中的独特表达模式取决于在整合位点内头对头排列的两个转基因的回文排列。由于启动子接合区的缺失，DNA序列的少量丢失会废除基因表达。 我们已经开发了一种检测这些缺失事件的方法，并将该方法应用于Tg.AC育种者的基因型筛选。 为了研究回文区在转基因表达中的作用，我们表征了Tg.AC x FVB/N异交中天然存在的突变体的突变频率。突变频率确定为1.5+/-2%。 利用Tar克隆技术，我们克隆了整合位点和转基因，并将克隆重新导入FVB/N癌细胞系。转基因表达在所有细胞中是明显的，无论侧翼的DNA含量，暗示区域内的转基因整合位点和回文区的作用一致。 DNA酶I足迹和凝胶移位实验表明多个转录因子结合位点集中在回文对称轴附近以及基础启动子区。 这些因子也存在于白血病细胞中，其也表达转基因，但不存在于正常的非转基因表达皮肤中。 最后，我们已经完成了我们的第一个基因阵列实验，研究基因表达的变化之前和之后的转基因激活。这项工作表明，当消除毛囊周期差异时，Tg.AC和FVB/N小鼠之间的基因表达变化很少。

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.