Identification of Novel Tumor Suppressor Genes in Breast Cancer Using Gene Tapping Technique
Final rept. 30 Sep 2000-30 Sep 2001
COLORADO STATE UNIV FORT COLLINS
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Cancer is a multi-step process resulting from a number of genetic changes in cells. The role of tumor suppressor genes in breast carcinogenesis, especially at the early stage, remains largely unknown. We hypothesize that during the transformation of a breast epithelial cell, loss of function of several yet unidentified genes tumor suppressors results in either a partially or fully transformed phenotype. The aim of this study is to identify novel tumor suppressor genes involved in breast epithelial transformation using the gene-trapping technique. We used the polyA-trap retroviral vector pRET for infection into non-tumorigenic human mammary epithelial cells MCF 1 0A. We screened for clones where functional genes have been trapped by selection for G4 18-resistance. We essentially established a gene-trapped library of MCF 1 0A clones where expression of a single gene per clone is disrupted. We screened for transformed clones using the soft-agar cloning assay for anchorage independent growth. We isolated 25 transformed clones and identified the trapped genes in 5 clones by rapid amplification of cDNA ends 3 RACE. We identified 2 known genes and 2 novel genes as putative tumor suppressor genes. Further characterization of these genes will elucidate their role in the early transformation process in breast epithelial cells.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research