MICRO QUANTITY CDNA LIBRARIES--BREAST TUMOR EXPRESSION

  • Beisel, Kirk (PI)

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date9/30/979/29/01

Funding

  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute: $437.00
  • National Cancer Institute

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