MICRO QUANTITY CDNA LIBRARIES--BREAST TUMOR EXPRESSION

描述：（申请人的描述） 为了开发乳腺肿瘤中表达基因的完整索引， 人乳腺癌全长cDNA文库的构建 组织是重要的第一步。 为建设初步有关 cDNA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的可用性 恶性进展，如非典型增生，原位癌， 原发性侵袭性肿瘤和转移性疾病，不与 正常或干扰细胞群，和（2）分子方法学 按比例缩小以使用含有完整的 mRNA。 我们建议将重点放在Microquantity-cDNA的标准化上 （Mq-cDNA）文库构建和确定其用于 人类乳腺细胞的表达分析，并将分三个阶段进行。 第一阶段将是优化我们目前的技术， 通过构建单向Mq-cDNA使用该方法 使用MCF 7和MDA 435乳腺肿瘤细胞系构建文库。 我们将 通过鉴定最有效和最有效的方法获得全长序列， 包括5'末端的方案。 将进行研究，以优化和 保持成绩单的表达和频率。 最佳条件 将确定扩增的效率和重复使用的效率 将测试ds-cDNA模板。 第二阶段将利用 用于建立正常和肿瘤性乳腺文库的MQ-cDNA方案 细胞，以确定这种方法的可行性和有效性。 将构建代表乳腺癌的Mq-cDNA模板和文库， 来自原发性和转移性非癌乳腺细胞的组织来源细胞 乳腺细胞和培养物。 第三阶段是利用Mq-cDNA 快速鉴定肿瘤相关基因表达。 所得到的Mq-cDNA模板和文库将用于研究基因的表达。 在乳腺肿瘤中的表达。 将在1）静息与 非恶性乳腺上皮的增殖群体，2）恶性 和来源于其的非恶性乳腺上皮培养物 个体，和3）来自淋巴结阴性的病例匹配肿瘤 （“非侵袭性”肿瘤）与淋巴结阳性（“侵袭性”肿瘤） 患者 对于这些比较， DDRT-PCR和抑制性消减杂交 PCR将用于鉴定差异基因表达。

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.