Chraracterization of follicular stem cells in Tg.AC mice

点击翻译按钮获取中文摘要

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.