Rodríguez, J.A. and Henderson, B.R.
Notes: Several cancer-associated proteins, such as the tumor suppressor p53 and the oncoproteins c-ABL and hdm2, have been shown to shuttle between the nucleus and the cytoplasm. Due to this information and the controversy regarding BRCA1 localization, the authors wanted to determine whether BRCA1 is also capable of nuclear-cytoplasmic shuttling.
To this end, a plasmid encoding untagged BRCA1 was created by subcloning the full-length BRCA1 cDNA into a vector as a NotI/ClaI fragment. To generate pYFP-BRCA1, a DNA fragment encoding the yellow fluorescent protein (YFP) was amplified by polymerase chain reaction (PCR) and inserted in frame at the end of the BRCA1 cDNA in the plasmid, using the unique NotI restriction site.
Human breast cancer cell lines T47D and MCF-7, the human breast epithelial cell line HBL-100, and mouse NIH3T3 fibroblasts were seeded onto glass coverslips and transfected at 50–70% confluency with 0.5–2µg of plasmid DNA using the FuGENE® 6 Transfection Reagent. After 48 hours, cells were fixed and processed for fluorescence microscopy. (4283)