Susceptibility of BRCA2 Heterozygous Normal Mammary Epithelial Cells to Radiation-Induced Transformation
Annual rept. 17 Sep 2004-16 Sep 2005
EVANSTON NORTHWESTERN HEALTHCARE RESEARCH INST IL
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In the approved Statement of Work, we proposed six tasks for the first two years. Task3 turned out to be much more difficult than we have anticipated. Thus far we have not yet obtained clones with one allele of BRCA2 disrupted even after numerous attempts. In last year s reported we proposed three alternative strategies. We have performed all three sets of experiments. We constructed the promoter less constructs to target exon11. We also constructed the promoter less constructs to target exon2. However, we have failed to obtain BRCA2- cell lines. Our alternative strategy, the RNAi strategy has been successful. We already obtained stable cell lines with reduced levels of BRCA2 expression. Since RNAi does not ideally mimic the in vivo situation, we will use these cell lines as a backup. We have preliminary data indicated that cell surface marker such as CD19 and Hook-2 can be used as efficient selection markers using magnetic beads. We are currently in the process of generating knock-out construct s using CD19 and Hook-2 as selection markers. Once BRCA2-cell lines are generated, the remaining task of the project will be carried out.
- Medicine and Medical Research