CHARACTERIZING MURINE GI STEM CELLS: NEW MOLECULAR TOOLS

描述（由申请人提供）：干细胞（sc）几乎肯定是肠癌的起源细胞。但是，由于没有特异性的标记物来识别SO，因此SC的机制是模糊的。我们的长期目标是使用动物模型来开发和测试用于在体内识别成人肠道sc的研究工具。在这里，我们将测试一种工具，GFP-Tcf 4转基因小鼠。我们设计了这种小鼠系，当Tcf 4被激活时，该小鼠系显示绿色荧光蛋白阳性（GFP+）细胞。Tcf 4是重要的，因为1）肠道肿瘤已激活Tcf 4由于致癌的Apc突变和2）现有的证据表明，Tcf 4在正常的隐窝激活是独特的sc。因此，标记Tcf 4激活可能是一个标记sc。我们交叉GFP-Tcf 4小鼠与ApcMin小鼠，因为后者是在高风险的肠癌和评估Apc突变对Tcf 4激活的影响。目的1：鉴定、分离和鉴定GFP-Tcf 4小鼠的GFP+肠上皮细胞。这些小鼠中的GFP+肠细胞似乎定位于隐窝基底（SC驻留的地方）。为了量化这一发现，我们将a）使用荧光显微镜/高级图像分析（肠切片）测量体内GFP信号强度，沿着隐窝轴，和B）流式细胞术分选（分离的固定的GFP+肠细胞，其源自纯化的整个隐窝）。假设1（H1）：GFP+肠细胞仅在隐窝基底中，是一小部分隐窝细胞，对核β-连环蛋白染色（（c））。AIM 2.为了观察，使用如上所述的显微镜和细胞计量术，GFP报告基因是否对Tcf 4激活的变化做出响应。H2：GFP+的隐窝细胞的数量&显示核定位在突变隐窝中增加（正常外观的粘膜[1个Apc突变]，腺瘤性粘膜[2个突变]）。AIM 3.分离活的GFP+和GFP-肠细胞。在这里，我们将分离活的（未固定的）肠上皮细胞并通过流式细胞术对其进行分选。H3：防止肠上皮细胞的单细胞悬浮液中的凝集将允许活细胞的流式细胞术分选; GFP+而不是GFP-细胞将显示核染色& Hoechst染料流出（一种功能性SC特性）测试阳性。意义如果预测成立，目标1结果将表明GFP表达ID的肠SC（或SC样细胞）;目标2结果，报告分子响应Tcf 4活化的变化;目标3结果，活的GFP+肠细胞是可分离的和可表征的，因此可以测试它们是否具有其他SC特性。然后可以评估SC在肠道肿瘤的发生和促进中的作用。

DESCRIPTION (provided by applicant): Stem cells (sc) are almost certainly the cells of origin of intestinal cancer. But, sc mechanisms are obscure because there are no specific markers to identify SO. Our long-term objective is to use animal models to develop & test research tools for identifying adult intestinal sc in vivo. Here we will test one tool, GFP-Tcf4 transgenic mice. We designed this mouse line to display green fluorescence protein positive (GFP+) cells when Tcf4 is activated. Tcf4 is important because 1) intestinal tumors have activated Tcf4 due to cancer-causing Apc mutations & 2) available evidence suggests that Tcf4 activation in normal crypts is unique to sc. Thus, a marker for Tcf4 activation may be a marker for sc. We crossed GFP-Tcf4 mice with ApcMin mice because the latter are at high risk for intestinal cancer & to assess effects of Apc mutations on Tcf4 activation. AIM 1: To identify (ID), isolate & characterize GFP+ enterocytes from GFP-Tcf4 mice. GFP+ enterocytes in these mice appear localized to the crypt base (where SC reside). To quantify this finding, we will a) use fluorescence microscopy/advanced image analysis (of intestinal sections) measuring in vivo GFP signal intensity along the crypt axis, & b) flow cytometry sorting (of isolated fixed, GFP+ enterocytes derived from purified whole crypts). Hypothesis 1(H1): GFP+ enterocytes are only in the crypt base, are a small % of crypt cells, stain for nuclear (-catenin ((c). AIM 2. To see, using microscopy & cytometry as above, if the GFP reporter responds to changes in Tcf4 activation. H2: The # of crypt cells that are GFP+ & show nuclear (c localization are increased in mutant crypts (normal-appearing mucosa [1 Apc mutation], adenomatous mucosa [2 mutations]). AIM 3. To isolate viable GFP+ & GFP- enterocytes. Here we will isolate viable (not fixed) enterocytes & sort them by flow cytometry. H3: Prevention of clumping in single cell suspensions of enterocytes will permit flow cytometric sorting of viable cells; GFP+ but not GFP- cells will show nuclear (c staining & test positive for Hoechst dye efflux, a functional SC property. Significance. If predictions hold, Aim 1 results will suggest that GFP expression ID's intestinal SC (or sc-like cells); Aim 2 results, that the reporter responds to changes in Tcf4 activation; Aim 3 results, that viable GFP+ intestinal cells are isolatable and characterizable hence can be tested to see if they have other sc properties. One could then evaluate the role of SC in initiation & promotion of intestinal neoplasms.