Regulation of Transgene Expression

美国国家毒理学计划(NTP)正在对Tg.AC小鼠模型进行评估，作为常规两年生物测试的补充。短期26周的生物测试依赖于Tg.AC转基因小鼠在局部使用化学致癌物治疗时会产生皮肤乳头状瘤的经验观察。在FVB/N鼠标上创建的五条方块线中只有一条显示了现在与Tg.AC鼠标相关的独特特征。该实验室的研究目标是了解Tg.AC小鼠诱导的致瘤反应以及对v-Ha-ras转基因的依赖。转基因被认为是异位表达的，因为有染色体整合位点。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整合部位内两个转基因头对头排列的回文排列。DNA序列的少量丢失，由于启动子连接区的缺失，导致基因表达受阻。我们已经开发了一种检测这些缺失事件的方法，并将这种方法应用到Tg.AC育种者的基因筛查中。为了研究回文区域在转基因表达中的作用，我们对Tg.AC×FVB/N OUT杂交中自然发生的突变频率进行了表征。突变频率为1.5+/-2%。利用焦油克隆技术克隆了整合位点和转基因，并将克隆重新导入FVB/N细胞系。无论DNA的侧翼含量如何，转基因在所有细胞中的表达都是明显的，这意味着转基因整合部位内有一个区域，并与回文区域的作用一致。DNase I足印和凝胶移动实验表明，多个转录因子结合位点集中在回文的对称轴附近以及基本的启动子区域。这些因子也存在于白血病细胞中，白血病细胞也表达转基因，但在正常的非转基因表达的皮肤中不存在。最后，我们完成了我们的第一个基因阵列实验，以研究转基因激活前后的基因表达变化。这项工作表明，消除毛囊周期差异后，Tg.AC和FVB/N小鼠之间的基因表达几乎没有明显变化。

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.