BRCA 1 FUNCTION USING AN INDUCIBLE TRANSGENE

携带BRCA1肿瘤抑制基因生殖系突变的女性一生中患乳腺癌的风险可能高达80%至90%。近年来，通过研究BRCA1蛋白的表达模式、细胞定位、分子间相互作用以及在转录激活和DNA损伤反应中的潜在作用，对BRCA1蛋白功能的认识取得了重大进展。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答案。这种理解上的差距尤其重要，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起着特定的作用。本研究旨在阐明BRCA1对小鼠乳腺上皮细胞增殖和分化的影响机制，具体研究内容如下：I.开发和表征转基因小鼠系统，允许以乳腺特异性方式诱导表达转基因。将生成转基因小鼠系统，以允许条件性乳腺上皮细胞特异性转基因表达、转基因表达水平的定量控制以及未诱导状态下的最小转基因表达。二.利用乳腺特异性、可诱导的转基因系统改变转基因小鼠中BRCA1的表达。目的I中描述的诱导系统将用于干扰体内BRCA 1表达。BRCA 1表达的下调将通过针对内源性Brca 1转录物的锤头状核酶的条件性表达介导。BRCA1表达将通过外源性BRCA1小基因的条件表达上调。三.确定BRCA1在体内乳腺上皮增殖中的作用。将分析BRCA1表达失调对乳腺上皮细胞区室增殖的影响。在BRCA 1表达上调和下调的情况下，将通过BrdU标记确定乳腺上皮的增殖率。将确定BRCA 1表达失调对导管形态和细胞周期分子表达的影响。四.确定BRCA1在体内乳腺上皮分化中的作用。 在BRCA1上调和下调后，将确定乳腺中正常分化程序的改变。 该分析将在分子水平上通过确定基因表达的时间模式来进行，所述基因的表达通常对特定分化阶段具有特异性。 此外，还将确定BRCA1表达改变对妊娠期小叶肺泡分化的形态学变化特征的影响。

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.