BRCA 1 FUNCTION USING AN INDUCIBLE TRANSGENE

携带BRCA1抑癌基因胚系突变的女性患乳腺癌的终生风险可能高达80%至90%。最近，通过对BRCA1蛋白的表达模式、细胞定位、分子相互作用以及在转录激活和DNA损伤反应中的潜在作用的研究，人们在了解BRCA1蛋白的功能方面取得了重大进展。尽管有这些进展，关于BRCA1在乳腺上皮细胞生物学中的作用的基本问题仍然没有得到回答。这一认识上的差距尤其重要，因为乳腺癌是遗传突变BRCA1等位基因的个体的主要表型。由于癌症通常被认为是一种细胞增殖和分化失调的疾病，我们假设BRCA1在乳腺上皮细胞的这些过程中发挥着特殊的作用。为了阐明BRCA1对小鼠乳腺上皮细胞增殖和分化的影响机制，我们提出了以下具体目标：1.建立并鉴定一种转基因小鼠系统，该系统允许以乳腺特异的方式诱导转基因表达。将产生一个转基因小鼠系统，以允许有条件的乳腺上皮细胞特异性转基因表达，定量控制转基因表达水平，并在未诱导状态下最小限度地表达转基因。使用乳腺特异的、可诱导的转基因系统改变转基因小鼠中BRCA1的表达。AIM I中描述的诱导系统将被用来干扰BRCA1在体内的表达。BRCA1表达的下调将通过针对内源性BRCA1转录本的锤头状核酶的有条件表达来介导。外源BRCA1微型基因的条件表达将上调BRCA1的表达。确定BRCA1在体内乳腺上皮细胞增殖中的作用。我们将分析BRCA1表达失调对乳腺上皮细胞间增殖的影响。在BRCA1表达上调和下调的情况下，乳腺上皮细胞的增殖率将通过BrdU标记来确定。BRCA1表达异常对导管形态和细胞周期分子表达的影响将被确定。确定BRCA1在体内乳腺上皮细胞分化中的作用。当BRCA1上调和下调时，乳腺正常分化程序的变化将被确定。这种分析将在分子水平上进行，方法是确定基因表达的时间模式，这些基因的表达通常是特定分化阶段的。此外，BRCA1表达的改变对妊娠期间小叶肺泡分化特征的形态变化的影响将被确定。

Women carrying germline mutations in the BRCA1 tumor suppressor gene have a lifetime risk of breast cancer that may be as high as 80 to 90%. Recently, significant progress has been made in understanding the function of the BRCA1 protein through study of its expression patterns, cellular localization, molecular interactions, and potential roles in transcriptional activation and DNA damage response. Despite these advances, fundamental questions regarding the role of BRCA1 in mammary epithelial cell biology remain unanswered. This gap in understanding is especially important since breast cancer is the predominant phenotype in individuals inheriting a mutant BRCA1 allele. As cancer is generally thought to be a disease of dysregulated cellular proliferation and differentiation, we hypothesize that BRCA1 plays specific roles in these processes in mammary epithelial cells. We propose to elucidate the mechanisms by which BRCA1 impacts on proliferation and differentiation in the mammary epithelium of the mouse by addressing the following specific aims: I. Develop and characterize a transgenic mouse system that allows inducible expression of transgenes in a mammary-specific manner. A transgenic mouse system will be generated to permit conditional mammary epithelial cell-specific transgene expression, quantitative control of transgene expression levels, and minimal transgene expression in the uninduced state. II. Use mammary-specific, inducible transgenic system to alter BRCA1 expression in transgenic mice. The inducible system described in aim I will be utilized to perturb BRCA1 expression in vivo. Downregulation of BRCA1 expression will be mediated by conditional expression of a hammerhead ribozyme directed against the endogenous Brca1 transcript. BRCA1 expression will be upregulated via conditional expression of an exogenous BRCA1 minigene. III. Determine the role of BRCA1 in mammary epithelial proliferation in vivo. The impact of dysregulated BRCA1 expression on proliferation in the mammary epithelial cell compartment will be analyzed. In the setting of both upregulated and downregulated BRCA1 expression, proliferation rates in the mammary epithelium will be determined by BrdU labeling. The effects of dysregulated BRCA1 expression on ductal morphology and expression of cell cycle molecules will be determined. IV. Determine the role of BRCA1 in mammary epithelial differentiation in vivo. Upon upregulation and downregulation of BRCA1, alterations in the normal differentiation programs in the mammary gland will be determined. This analysis will be performed at the molecular level by determining the temporal pattern of expression of genes whose expression is normally specific for particular stages of differentiation. In addition the effects of altered BRCA1 expression on the morphologic changes characteristic of lobuloalveolar differentiation in pregnancy will be determined.