MICRO QUANTITY CDNA LIBRARIES--BREAST TUMOR EXPRESSION

描述：(申请人描述) 为了建立在乳腺肿瘤中表达的基因的完整索引， 人乳腺全长代表性cDNA文库的构建 组织是必不可少的第一步。对于初步相关的建设 对于文库，必须满足两个重要的先决条件：(1) 代表不同阶段的同质细胞群体的可用性 恶性进展，如不典型增生、原位癌、 原发侵袭性肿瘤和未混合的转移性疾病 正常或干扰细胞群体，以及(2)分子方法学 缩减规模以使用少量包含完整的人类细胞 MRNA.我们建议将重点放在微量-cDNA的标准化上 (MQ-cDNA)文库的构建及其适用性的确定 对人类乳房细胞的表达分析，将分三个阶段进行。 第一阶段将优化我们现有的技术，以广泛应用于 该方法在单向MQ-cDNAs构建中的应用 文库采用MCF7和MDA435乳腺肿瘤细胞系。我们会 通过识别最有效和最有效的序列获得全长序列 包括5‘端的协议。将进行研究，以优化和 保持文字记录的代表性和频率。最优条件 用于扩增的效率将被确定和重复使用 将对DS-cDNA模板进行测试。第二阶段将利用 建立正常和肿瘤乳腺文库的MQ-cDNA法 以确定这一方法的可行性和有效性。 将构建代表乳房的MQ-cDNA模板和文库 原代和转移性非癌乳腺细胞的组织来源细胞 乳房细胞和培养。第三阶段是利用mq-cdna 文库在快速鉴定肿瘤相关基因表达中的作用。 所得到的mq-cDNA模板和文库将用于基因研究 在乳腺肿瘤中的表达。我们将在1)休息和休息之间进行比较 非恶性乳腺上皮的增殖群，2)恶性 和非恶性乳腺上皮细胞培养源自相同的 个体，以及3)病例匹配来自淋巴结阴性的肿瘤 (“非侵袭性”肿瘤)与淋巴结阳性(“侵袭性”肿瘤) 病人。对于这些比较，差异显示-反转 转录酶-聚合酶链式反应(DDRT-PCR)和抑制性消减杂交 聚合酶链式反应将被用来鉴定差异基因表达。

DESCRIPTION: (Applicant's Description) To develop a complete index of genes expressed in breast tumors, the construction of full length representational cDNA libraries of human breast tissue is an essential first step. For the construction of initial relevant cDNA libraries, two important prerequisites must be met: (1) the availability of homogeneous cell populations representing various stages of malignant progression, such as, atypical hyperplasia, in situ carcinoma, primary invasive tumor, and metastatic disease which are not admixed with normal or interfering cell populations, and (2) the molecular methodology scaled down to employ small populations of human cells containing intact mRNA. We propose to focus on the standardization of Microquantity-cDNA (Mq-cDNA) library construction and in determining its applicability for expression analyses of human breast cells, and will be done in three phases. The first phase will be to optimize our present technologies for widespread usage of this methodology by construction of unidirectional Mq-cDNA libraries using the MCF7 and MDA 435 breast tumor cell lines. We will obtain full length sequences by identifying the most efficient and effective protocol for including 5' ends. Studies will be done to optimize and maintain transcript representation and frequency. The optimal conditions for amplification will be determined and the efficiency of repeated use of ds-cDNA templates will be tested. The second phase will be to utilize the Mq-cDNA protocol for establishing libraries of normal and neoplastic breast cells in order to determine the feasibility and validity of this approach. Mq-cDNA templates and libraries will be constructed that represent breast tissue-derived cells from non-cancerous breast cells, primary and metastatic breast cells and cultures. The third phase will be to utilize Mq-cDNA libraries in the rapid identification of tumor-associated gene expression. The resulting Mq-cDNA templates and libraries will be used to study gene expression in breast tumors. Comparisons will be done between 1) resting vs proliferative populations of non-malignant breast epithelium, 2) malignant and non-malignant breast epithelial cultures derived from the same individual, and 3) case matched tumors from lymph node negative ("non-aggressive" tumors) versus lymph node positive ("aggressive" tumors) patients. For these comparisons both differential display-revers transcriptase-PCR (DDRT-PCR) and suppressive subtractive hybridization (SSH) PCR will be used to identify differential gene expression.