Chraracterization of follicular stem cells in Tg.AC mice

已显示v-Ha-ras Tg.AC小鼠 对特定化学物质产生反应而产生皮肤肿瘤， 全层创伤、紫外线照射和肿瘤 发展依赖于诱导表达的 转基因。以前的工作已经证明， 可在增生毛囊的区域中检测到表达 被一些人认为是一个假定的干细胞库。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 使用RT-PCR从少至50个细胞进行转基因表达。我们 建议使用这项技术来识别和表征 在最早时间表达转基因的细胞群 经过化学处理后。我们正在 开发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 与LCM，随后通过PCR扩增特定基因，以获得 深入了解基因表达定位于特定区域的正常 表皮毛囊和肿瘤。到目前为止，我们已经证明了 将生物素化的Oligo dT探针掺入细胞中， 免疫组化检测另一项技术， 发展结合免疫组织化学定位的一个 特定蛋白质（例如，p53）和LCM的阳性细胞， 特定基因的DNA提取和PCR扩增 用于序列分析。虽然我们主要使用 用于表征对克立西定中p53阳性细胞的技术 诱导膀胱肿瘤，我们预计，我们将能够使用 免疫组织化学检测人乳腺癌组织中cDNA掺入 结合LCM和基因表达分析。一个具体 我们想用这些技术解决的问题是什么 表皮干细胞所具有的特性， 转录激活的微环境 由造血启动子驱动的转基因。我们亦建议 使用这些技术作为比较基因表达的手段， 两阶段致癌模型引起的肿瘤之间 (i.e., FVB/N和/或FVB/N中的DMBA引发和DMBA促进 SENCAR小鼠）和在TPA促进的Tg中发育的那些小鼠。 小鼠

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.