This core will develop and maintain tools for the detection of gene products and manipulation of genes thought to play roles in the responses of human breast tumor cells to therapeutic drugs. Preliminary assessment of endogenous gene states will be performed using the comparative and computational techniques described in Projects 1-3. Validated oligonucleotide primer sets, DNA probes, antibodies, and fluorescence-based assays will be used for relative quantitation of the products and functions of endogenous genes. Directly determining the functional contribution of individual genes to given phenotypes will require manipulation of the genes in cellular systems under conditions where extraneous variables are minimized. Both full-length open reading frame cDNAs and DNAs encoding shRNAs will therefore be employed for up- and down-regulation of individual genes in cultured HMEC and breast tumor cell lines. Where appropriate, cDNAs encoding dominant active protein mutants will be used to mimic specific tumor cell defects. Efficient uptake and stable expression of gene constructs will be facilitated through the use of retroviral vectors bearing drug resistance markers (e.g., neo r, puro r, hygro r) for positive selection. Tasks performed by the core will be divided into three categories: 1) acquisition of antibodies, cDNAs, and shRNAs, and subcloning the latter, if necessary, into suitable expression vectors, 2) growth, isolation and testing of plasmid, retrovirus, and antibody stocks, 3) storage and management of the acquired or generated reagents. These tasks will be performed in a centralized location by an experienced full-time research associate under the direction of two staff scientists.
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