Chraracterization of follicular stem cells in Tg.AC mice

V-Ha-ras Tg.AC小鼠已被证明 对特定的化学物质产生皮肤肿瘤， 全身创伤、紫外线照射和肿瘤 发育依赖于诱导表达 转基因。先前的研究已经证明，转基因 可在增生性毛囊区域检测到表达 被一些人确认为假定的干细胞储存库。我们有 证明了我们可以成功地从组织中分离出RNA 进行激光捕获显微切割(LCM)，然后检测 利用RT-PCR从50个细胞中获得转基因表达。我们 建议使用这项技术来识别和表征 最早表达转基因的细胞群体 化学处理后的穴位。我们正在进行 开发有可能加速实现这一目标的技术。一 正在开发的技术是结合原位cdna合成 用LCM，然后用PCR扩增特定基因以获得 对基因表达的洞察局限于正常的特定区域 表皮、毛囊和肿瘤。到目前为止，我们已经演示了 将生物素标记的寡聚脱氧核糖核酸探针掺入细胞 免疫组织化学检测。另一种技术在 发展结合免疫组织化学定位的一个 阳性细胞的特异性蛋白(如P53)和LCM 特定基因的DNA提取和聚合酶链式反应扩增 用于序列分析。尽管我们主要使用的是这个 P-Csidine中P53阳性细胞的鉴定技术 诱导的膀胱肿瘤，我们预计我们将能够使用 肝细胞癌组织中c DNA掺入的免疫组织化学检测 结合激光共聚焦显微镜和基因表达分析。一个特定的 我们希望使用这些技术解决的问题是 表皮干细胞所具有的特征提供了 转录激活的允许微环境 由造血剂驱动的转基因。我们还建议 使用这些技术作为比较基因表达的一种手段 两阶段致癌模型所致肿瘤之间的关系 (即，在FVB/N和/或中的DMBA启动和DMBA推广 Sencar小鼠)和在TPA启动子Tg.AC中发育的 老鼠。

The v-Ha-ras Tg.AC mouse has been shown to develop cutaneous neoplasms in response to specific chemicals, full-thickness wounding, and UV radiation exposure, and tumor development is dependent upon induction of expression of the transgene. Previous work has demonstrated that transgene expression can be detected in a region of hyperplastic hair follicles identified by some as a putative stem cell reservoir. We have demonstrated that we can successfully isolate RNA from tissue subjected to Laser Capture Microdissection (LCM) and then detect transgene expression using RT-PCR from as few as 50 cells. We propose to use this technology to identify and characterize the population of cells expressing the transgene at the earliest time point following chemical treatment. We are in the process of developing techniques that will potentially expedite that goal. One technique under development is combining in situ cDNA synthesis with LCM, followed by PCR amplification of specific genes to gain an insight into gene expression localized to specific areas of normal epidermis, hair follicles, and tumors. We have thus far demonstrated incorporation of a biotinylated Oligo dT probe into cells using immunohistochemical detection. Another technique under development combines immunohistochemical localization of a specific protein (e.g., p53) and LCM of positive cells followed by DNA extraction and PCR amplification of a specific gene or genes for sequence analysis. Although we have primarily used this technique for characterizing p53-positive cells in p-cresidine induced bladder tumors, we anticipate that we will be able to use immunohistochemical detection of cDNA incorporation in combination with LCM and gene expression analysis. One specific question we would like to address using these techniques is what characteristics epidermal stem cells possess that provide a permissive microenvironment for transcriptional activation of a transgene driven by a hematopoietic promoter. We also propose to use these techniques as a means of comparing gene expression between neoplasias arising from two-stage carcinogenesis models (i.e., DMBA initiation and DMBA promotion in FVB/N and/or SENCAR mice) and those developing in TPA-promoted Tg.AC mice.